CN112437674A

CN112437674A - Lipid prodrugs for drug delivery

Info

Publication number: CN112437674A
Application number: CN201980039677.7A
Authority: CN
Inventors: 迈克尔安·塔尔蒂斯; 利利娅·福劳罗娃
Original assignee: Research Park Co Of New Mexico University Of Science And Technology
Current assignee: Research Park Co Of New Mexico University Of Science And Technology
Priority date: 2018-04-11
Filing date: 2019-04-11
Publication date: 2021-03-02
Also published as: WO2019200043A1; JP2021521279A; EP3773734A4; US20210361575A1; EP3773734A1

Abstract

The present disclosure describes the synthesis and use of lipid prodrugs that self-assemble into lipid microbubbles or liposomes. The prodrug-loaded microvesicles or liposomes can be activated intracellularly using an external stimulus, for example using ultrasound.

Description

Lipid prodrugs for drug delivery

Cross-referencing

This application claims the benefit of U.S. provisional application No. 62/656,035 filed on 11/4/2018, the entire contents of which are incorporated herein by reference.

Government rights

The invention was made with U.S. government support under approval numbers P20 GM103451 and P20 RR016480 awarded by the National Institutes of Health.

Background

Natural and synthetic chemotherapeutic agents are often not amenable to laboratory and clinical trials due to poor water solubility, instability, insufficient site specificity, general toxicity or formulation problems. Liposomes are spherical vesicles having at least one lipid bilayer. Liposomes can be used as vehicles for administering nutrients and drugs. Bioavailability and site specificity of drugs can be improved by liposome-mediated drug delivery.

Is incorporated by reference

Each of the patents, publications, and non-patent documents cited in this application is incorporated by reference herein in its entirety as if each were individually incorporated by reference.

Disclosure of Invention

In some embodiments, the present disclosure provides a lipid-based carrier comprising: a) a surface layer, wherein the surface layer comprises a prodrug, wherein the prodrug comprises a therapeutic agent covalently conjugated to a phospholipid; and b) a core, wherein the surface layer surrounds the core.

In some embodiments, the present disclosure provides a method of treating a disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a lipid-based carrier comprising: a) a surface layer, wherein the surface layer comprises a prodrug, wherein the prodrug comprises a therapeutic agent covalently conjugated to a phospholipid; and b) a core, wherein the surface layer surrounds the core.

Drawings

Figure 1 shows a scheme in which cytarabine may be conjugated to a phospholipid.

Figure 2 panel a shows the synthesis of prodrugs, self-assembly of prodrug-loaded liposomes, and the use of liposomes for treating cells in vitro. Panel B shows the use of prodrug-loaded microbubbles and ultrasound exposure for targeted drug delivery in vitro.

Figure 3 shows a schematic of microbubble therapy using ultrasound as an in vitro trigger.

Figure 4 shows the synthetic pathway for coupling cytarabine and topotecan to phospholipids.

Figure 5 shows the differential scanning calorimetry curve of 2T-P loaded liposomes at increasing concentrations.

Figure 6 shows the differential scanning calorimetry curve of increasing concentrations of P-loaded liposomes.

Figure 7 panel a shows drug incorporation in liposomes before and after extrusion for 2T-P. Panel B shows drug incorporation in liposomes before and after extrusion for 2T-N.

Figure 8 panel a shows that 2T-P loaded liposomes remained stable for the entire 3 week period. Panel B shows that the P-loaded liposomes were stable over the entire 3 week period. Panel C shows that 2T-N loaded liposomes remained stable for the entire 3 week period. Panel D shows that the N-loaded liposomes remained stable for the entire 3 week period.

Figure 9 shows drug incorporation in liposomes before and after 2T-T extrusion.

FIG. 10 shows size distribution curves for liposomes with varying amounts of 2T-T before (pre-ex) and after (post-ex) extrusion.

Figure 11 shows the mean diameter of the liposome population with varying amounts of 2T-T before and after extrusion (see figure 10).

Figure 12 shows the size distribution curves of liposomes with varying amounts of 2T-C before (pre) and after (post) extrusion.

Figure 13 shows the mean diameter of the liposome population with varying amounts of 2T-C before and after extrusion (see figure 12).

Figure 14 shows cytotoxicity of 2T-T loaded liposomes versus free T (topotecan).

FIG. 15 shows cytotoxicity of 2T-C loaded liposomes versus toxicity of free C (cytarabine).

Figure 16 shows ultrasound-triggered delivery of prodrug-loaded microbubbles in vitro.

FIG. 17 shows the fluorescence spectra of 2T-N loaded liposomes at different time points with and without treatment with pig liver esterase.

FIG. 18 shows the fluorescence spectra of empty liposomes at different time points with and without treatment with pig liver esterase.

FIG. 19 shows fluorescence spectra of PBS solutions at various time points with and without treatment with pig liver esterase.

Detailed Description

Many promising natural and synthetic chemotherapeutic agents are often not amenable to laboratory and clinical trials due to poor water solubility, instability, insufficient site specificity, general toxicity or formulation problems. Lipid-based carriers, such as liposomes, nanodroplets or microbubbles, can be used as vehicles for administering nutrients and drugs, and can significantly improve the bioavailability and site specificity of therapeutic agents.

Liposomes are spherical vesicles having at least one lipid bilayer. Liposomes have a core (e.g., an aqueous core) surrounded by a hydrophobic membrane in the form of a lipid bilayer. The main types of liposomes are multilamellar vesicles (MLVs) with several lamellar phase lipid bilayers, small unilamellar liposome vesicles (SUVs) with one lipid bilayer, Large Unilamellar Vesicles (LUVs) and helical vesicles. To deliver molecules (e.g., therapeutic agents) to the site of action, the lipid bilayer of the liposome can be fused with other bilayers, such as cell membranes. Liposomes can be used as carriers for dietary or nutritional supplements, or for targeted drug delivery.

Microbubbles are small gas-filled bubbles, typically between 0.5 μm and 10 μm in diameter. The core of the microbubble is gaseous, surrounded by a shell composed of, for example, polymers, lipids, lipopolymers, proteins, surfactants, or combinations thereof. Microbubbles are used as contrast agents in medical imaging and as carriers for targeted drug delivery. Microbubbles resonate strongly at high frequencies when used in ultrasound scanning and reflect megasonic waves more efficiently than body tissue. Microbubbles are approximately the same size as red blood cells, exhibit similar rheological properties in blood vessels, and can be used to measure blood flow in organs or tumors.

Nano-droplets are small, liquid-filled bubbles, smaller than microbubbles. In some embodiments, the shell of the nanodroplets comprises, for example, a lipid or a phospholipid. The nano-droplets may be filled with a liquid that is easily evaporated. As the liquid core of the nano-droplets evaporates, the nano-droplets transform into microbubbles. Non-limiting examples of liquid cores for use in the nanodroplets include perfluorocarbons and perfluorobutanes.

The present disclosure describes the development and use of a group of lipid prodrugs, the molecular interactions of lipid prodrugs within lipid-based carriers and the efficacy of lipid prodrugs in vivo and in vitro using ultrasound. Non-limiting examples of lipid-based carriers include liposomes and microbubbles. The synthetic strategies described herein allow for loading effective lipid prodrugs into lipid-based carriers (e.g., liposomes and microbubbles) to form prodrug-loaded lipid-based carriers (PLLBCs). In some embodiments, PLLBC may be used as an ultrasound contrast agent and in combination with ultrasound for site-directed therapy that allows real-time visualization of a target (e.g., a malignancy) and the contrast agent to reach the target site.

Lipid prodrugs can utilize lipid-based drug carriers, targeted delivery strategies and ultrasound-mediated techniques to achieve better performance by: 1) increasing the drug payload; 2) purification and solubility problems are minimized by vehicle self-assembly; 3) preventing premature release of the drug from the vehicle; 4) keep activated by ultrasound imaging and therapeutic techniques that bring the drug in proximity to the target cells; and 5) maintenance of drug efficacy after rapid intracellular lysis. The conjugation of lipids and drugs via cleavable ester bonds in a sterically unhindered manner enables the drugs to self-assemble at high concentrations into carriers that are activated by ultrasound; and release effective and fast acting drugs after intracellular uptake.

The lipid prodrugs and methods described herein are useful for targeting tumors, such as unresectable pancreatic, liver, and brain tumors. In some embodiments, the prodrugs and methods described herein can be used to treat pancreatic cancer by targeting the stromal matrix of the subject. In some embodiments, the methods described herein can be used for drugs that have toxicity or solubility issues. In some embodiments, a targeting ligand may be added to the microvesicles described herein.

Lipid prodrugs

In some embodiments, the present disclosure describes lipid prodrugs that can first self-assemble into lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) and then remain inactive until activated within the cell after deposition by an external stimulus. In some embodiments, the synthetic methods described herein attach an activated phospholipid to a therapeutic agent having a sterically unhindered hydroxyl attachment site. This structure allows simple conjugation by esterification. In some embodiments, the synthetic methods described herein attach an activated phospholipid to a therapeutic agent having an sterically unhindered amine-based attachment site. This structure allows simple conjugation by amidation. After conjugation of the therapeutic agent to the activated phospholipid, enzymatic reaction cleavage can cause cleavage in a biological environment.

The present disclosure also describes methods of inserting drugs into lipid-based carriers. Non-limiting examples of lipid-based carriers include liposomes, nanodroplets, and microbubbles. In some embodiments, the present disclosure utilizes FDA approved drugs or drugs that are well characterized but are limited in clinical use due to solubility issues or extremely potent efficacy. To facilitate loading into lipid-based carriers, drugs can be attached to activated phospholipids to form prodrugs. The prodrug can then be incorporated into a lipid-based carrier by, for example, self-assembly to form PLLBC. PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can comprise a therapeutic agent, such as an antiviral agent, an antibacterial agent, an anti-cancer agent, a neurotransmitter, a protein, a dermatological agent, a cosmetic agent, a chelator, or a biological agent. In some embodiments, PLLBCs of the present disclosure include a combination of prodrugs. In some embodiments, PLLBCs of the present disclosure comprise a dye molecule.

PLLBCs of the present disclosure, such as liposomes, nanodroplets, or microbubbles, can surround the core material. Non-limiting examples of nuclear materials include gases, such as sulfur hexafluoride (SF)₆) Or perfluoropropane; solids such as titanium nitride, superparamagnetic iron oxide, gold, silver, iron, copper, zinc, titanium, platinum, gadolinium and palladium; semiconductor, exampleSuch as silicon dioxide; an inorganic material; an organic material; an aqueous solution; and liquids such as perfluorocarbons and perfluorobutanes.

PLLBCs of the present disclosure may comprise an antiviral agent. Antiviral drugs can minimize symptoms and infectivity and shorten the course of the disease. PLLBCs of the present disclosure may comprise an antiviral agent that inhibits attachment and penetration of a virus into a host cell, release of nucleic acids, replication of a viral genome, translation of viral mRNA, assembly of viral components, or release of a new virus from a host cell. In some embodiments, PLLBCs of the present disclosure may comprise an antiviral agent, such as amantadine, a nucleoside analog (e.g., acyclovir, ganciclovir, foscarnet), a nucleoside reverse transcriptase inhibitor (NRTI; e.g., lamivudine), a non-nucleoside reverse transcriptase inhibitor (e.g., nevirapine, efavirenz), interferon alpha, a protease inhibitor (e.g., boseprevine), or a neuraminidase inhibitor (e.g., oseltamivir).

In some embodiments, PLLBCs of the present disclosure may comprise an antiviral agent against influenza virus, such as an ion channel blocker (e.g., amantadine, rimantadine) or a neuraminidase inhibitor (e.g., oseltamivir or zanamivir). In some embodiments, PLLBCs of the present disclosure may comprise antiviral agents against herpes viruses, such as guanosine analogs (e.g., acyclovir, penciclovir, valacyclovir, famciclovir, ganciclovir, valganciclovir) or direct viral DNA polymerase inhibitors (e.g., foscarnet, cidofovir). In some embodiments, PLLBCs of the present disclosure may comprise antiviral agents against hepatitis b and c, such as nucleotide analogs (e.g., tenofovir, adefovir, lamivudine, entecavir, telbivudine), antiviral and immunomodulatory agents via intercellular and intracellular mechanisms (e.g., PEG-interferon- α), guanosine analogs (e.g., ribavirin), protease inhibitors (e.g., cimetivir), non-nucleoside polymerase (NS5A) inhibitors (e.g., ledipasvir, vipatavir), or non-nucleoside polymerase (NS5B) inhibitors (e.g., sofosbuvir).

PLLBCs of the present disclosure may comprise antibacterial agents, such as anilides, quinolones, sulfonamides, penicillins, protein synthesis inhibitors (e.g., macrolides, lincosamides, tetracyclines), biguanides, bisphenols, halophenols, phenols, cresols, or quaternary ammonium compounds. In some embodiments, a PLLBC of the present disclosure may comprise triclocarban, chlorhexidine, alexidine, polybiguanides, hexachlorophene, parachloro-metaxylenol (PCMX), phenol, cresol, cetrimide, benzalkonium chloride, norfloxacin, polymyxin B, oxacillin, dicloxacillin, tetracycline, vancomycin, penicillin, rifamycin, lipiarmycin (lipiarmycin), streptomycin, amphotericin B, cephalosporins, or cetylpyridinium chloride.

PLLBCs of the present disclosure may comprise an anti-cancer agent. Non-limiting examples of anti-cancer agents include polyfunctional alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, antibiotics, hormonal agents, or other anti-cancer agents. In some embodiments, PLLBCs of the present disclosure may comprise an anticancer agent, such as podophyllotoxin (P), 7- (3, 5-dibromophenyl) -2-hydroxy-7, 11-dihydrobenzo [ h []-furo [3,4-b]Quinolin-8 (10H) -one (N), cyclophosphamide, ifosfamide, mechlorethamine, melphalan

Chlorambucil (Leukeran)^TM) Thiotepa (thiopeta)

Busulfan medicine

Carmustine, lomustine, semustine, procarbazine

Dacarbazine (DTIC), altretamine

Cis-platinum

Methotrexate, mercaptopurine (6-MP), thioguanine (6-TG), fludarabine phosphate, cladribine

Pentostatin

Fluorouracil (5-FU), cytarabine (Ara-C), azacitidine, vinblastine

Vincristine

Etoposide (VP-16),

) Teniposide, teniposide

Topotecan

Irinotecan

Paclitaxel

Docetaxel

Anthracyclines (e.g., doxorubicin, daunorubicin), actinomycins

Idarubicin (Idarubicin)

Precamycin

Mitomycin

Bleomycin

Tamoxifen

Flutamide

Gonadotropin releasing hormone agonists (e.g., leuprorelin, goserelin), aromatase inhibitors (e.g., aminoglutethimide, anastrozole

Amsacrine, gemcitabine, melphelan, methotrexate, hydroxyurea

Asparaginase

Mitoxantrone

Mitotane, retinoic acid derivatives, bone marrow growth factor or amifostine (aminfosine).

PLLBCs of the present disclosure may comprise a neurotransmitter. Non-limiting examples of neurotransmitters include amino acids, gas transmitters, monoamines, trace amines, peptides, purines, or other neurotransmitters. In some embodiments, the liposomes, nanodroplets, or microbubbles of the present disclosure may comprise neurotransmitters, such as glutamic acid, aspartic acid, D-serine, gamma-aminobutyric acid (GABA), glycine, dopamine, norepinephrine, epinephrine, histamine, 5-hydroxytryptamine, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronine, octopamine, tryptamine, oxytocin, somatostatin, substance P, cocaine and amphetamine regulated transcripts, opioid peptides, Adenosine Triphosphate (ATP), adenosine, acetylcholine (ACh), and arachidonic acid ethanolamine.

PLLBCs of the present disclosure may comprise a protein or a biologic. Non-limiting examples of proteins or biologics include peptides; peptide fragments; antibodies such as bivalent antibodies, monovalent antibodies, polyclonal antibodies and monoclonal antibodies; an antibody fragment; and nanobodies.

Synthesis method

The present disclosure describes methods of synthesizing PLLBCs such as liposomes, nanodroplets, or microbubbles. The prodrug-loaded liposomes or microbubbles can comprise the therapeutic agent in the respective lipid layers. The prodrug-loaded liposomes of the present disclosure can comprise a therapeutic agent in the lipid bilayer of the liposome, and the prodrug-loaded microvesicles of the present disclosure can comprise a therapeutic agent in the lipid monolayer of the microvesicle.

In some embodiments, PLLBC can be produced by incorporating a lipid prodrug into the lipid shell of a lipid-based carrier. In some embodiments, incorporation of the lipid prodrug into the lipid shell of a lipid-based carrier can eliminate leakage, covalently bind the drug to the lipid-based carrier, and deliver a dual targeting strategy in one dose. In some embodiments, incorporation of a lipid prodrug into the lipid shell of a lipid-based carrier can affect the Pharmacokinetics (PK) or Pharmacodynamics (PD) of the drug.

In some embodiments, the prodrug is incorporated into the lipid shell of a lipid-based carrier using self-assembly. In some embodiments, incorporating the prodrug into the lipid shell of a lipid-based carrier can minimize or eliminate a purification step prior to administering the prodrug to a subject. In some embodiments, the prodrug-loaded microvesicles can be separated from the prodrug-loaded liposomes using centrifugation prior to administration. In some embodiments, incorporating the prodrug into the lipid shell of a lipid-based carrier can increase the amount of drug delivered to a target site within a subject while minimizing systemic dosing to the subject.

PLLBCs of the present disclosure (e.g., liposomes, nanodroplets, or microbubbles) can be assembled using solution prodrugs produced by conjugation of drug molecules or therapeutic agents to phospholipids. The drug molecule or therapeutic agent may be conjugated to an activated phospholipid, for example, a Maleimide (MAL) phospholipid, an activated carboxylic acid (NHS) -phospholipid, a glutaryl (Glu) -phospholipid, a 7-nitrophenyl-2-oxa-1, 3-oxadiazol-4-yl (NBD) -phospholipid, or a dithiopyridine (PDP) -phospholipid. In some embodiments, the drug molecule is conjugated to a MAL phospholipid, such as distearoyl N- (3-maleimide-1-oxopropyl) -L- α -phosphatidylethanolamine (DSPE-MAL); dimyristoyl N- (3-maleimide-1-oxopropyl) -L- α -phosphatidylethanolamine (DMPE-MAL); 1-palmitoyl-2-oleoyl N- (3-maleimido-1-oxopropyl) -L- α -phosphatidylethanolamine (POPE-MAL); or dipalmitoyl N- (3-maleimido-1-oxopropyl) -L-alpha-phosphatidylethanolamine (DPPE-MAL). In some embodiments, the drug molecule is conjugated to an NHS-phospholipid, such as distearoyl N- (succinimidyloxy-glutaryl) -L- α -phosphatidylethanolamine (DSPE-NHS); dioleoyl N- (succinimidyloxy-glutaryl) -L- α -phosphatidylethanolamine (DOPE-NHS); 1-palmitoyl-2-oleoyl N- (succinimidyloxy-glutaryl) -L- α -phosphatidylethanolamine (POPE-NHS); dipalmitoyl N- (succinimidyloxy-glutaryl) -L- α -phosphatidylethanolamine (DPPE-NHS); or dimyristoyl N- (succinimidyloxy-glutaryl) -L- α -phosphatidylethanolamine (DMPE-NHS).

In some embodiments, the drug molecule is conjugated to a Glu-phospholipid, such as distearoyl N-glutaryl-L- α -phosphatidylethanolamine (DSPE-Glu); dipalmitoyl N-glutaryl-L- α -phosphatidylethanolamine (DPPE-Glu); dimyristoyl N-glutaryl-L- α -phosphatidylethanolamine (DMPE-Glu); dioleoyl N-glutaryl-L- α -phosphatidylethanolamine (DOPE-Glu); or 1-palmitoyl-2-oleoyl N-glutaryl-L-alpha-phosphatidylethanolamine (POPE-Glu). In some embodiments, the drug molecule is conjugated to a PDP-phospholipid, such as dipalmitoyl N- [3- (2-pyridyldithio) -1-oxopropyl ] -L- α -phosphatidylethanolamine (DPPE-PDP).

Scheme 1 shows a non-limiting example of a synthesis scheme that can be used to produce prodrugs by synthesis of drug-conjugated phospholipids. In some embodiments, prodrugs are used to assemble PLLBCs of the present disclosure, such as liposomes, nanodroplets, or microbubbles. To produce a prodrug, a drug molecule or therapeutic agent comprising a hydroxyl, amino (amido), carboxyl, or sulfhydryl group is conjugated to an activated phospholipid in the presence of a dehydrating agent and a nucleophilic catalyst. In some embodiments, the activated phospholipid is a Glu-phospholipid, such as DPPE-Glu. In some embodiments, the dehydrating agent is N, N' -Dicyclohexylcarbodiimide (DCC) and the nucleophilic catalyst is 4-Dimethylaminopyridine (DMAP).

Scheme 1

The lipid prodrug may have a hydrophobic tail comprising saturated or unsaturated fatty acids. In some embodiments, the prodrug is double-tailed. In some embodiments, the prodrug molecule has a hydrophobic tail comprising a saturated fatty acid, which is a linear alkylene moiety. In some embodiments, the prodrug molecule has a hydrophobic tail comprising an unsaturated fatty acid that is a linear alkenylene moiety. In some embodiments, each R is¹And R²Is- (CH)₂)_n-, wherein n is from about 5 to about 24. In some embodiments, each R is¹And R²Is- (CH)₂)_n-, where n is 10. In some embodiments, each R is¹And R²Is- (CH)₂)_n-, where n is 12. In some embodiments, each R is¹And R²Is- (CH)₂)_n-, where n is 20.

Additional synthetic schemes may also be used to produce prodrugs by synthesizing drug-conjugated phospholipids. In some embodiments, a therapeutic agent, such as cytarabine, can be conjugated to a phospholipid via an amide or ester linkage, as shown in figure 1.

The prodrug solutions of the present disclosure can be used to assemble prodrug-loaded liposomes. Prodrug-loaded liposomes can be assembled by mixing a solution of drug-conjugated phospholipid (i.e., prodrug) with a phospholipid solution and a pegylated phospholipid solution and sonicating.

The prodrug solutions of the present disclosure can be used to assemble prodrug-loaded microbubbles. Prodrug-loaded microbubbles can be assembled by mixing and sonicating a solution of drug-conjugated phospholipid (i.e., prodrug) with a phospholipid solution and a pegylated phospholipid solution, and purging the resulting solution with a gas to form a gaseous core of the microbubble.

In some embodiments, the pegylated phospholipid solution used to assemble the drug-loaded lipid carrier (e.g., drug-loaded liposomes or drug-loaded microbubbles) includes, for example, DSPE-PEG (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) ammonium salt), DPPE-PEG ((N- (methylpolyoxyethylene oxycarbonyl) -1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) ammonium salt), DMPE-PEG (N- (methylpolyoxyethylene oxycarbonyl) -1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) ammonium salt), or any combination thereof. In some embodiments, pegylated phospholipid solutions used to assemble drug-loaded lipid carriers (e.g., drug-loaded liposomes or drug-loaded microbubbles) include, for example, DSPE-PEG2000(1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- (methoxy (polyethylene glycol 2000) ammonium salt), DSPE-PEG5000(1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 5000) ammonium salt), DSPE-PEG2000 carboxylic acid (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ carboxy (polyethylene glycol) -2000] (sodium salt)), DSPE-PEG5000 DBCO (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ dibenzocyclooctyl (polyethylene glycol) -5000] (ammonium salt)), DSPE-PEG5000 amine (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) -5000] (ammonium salt)), DSPE-PEG5000 maleimide (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -5000] (ammonium salt)), DSPE-PEG2000-TMS (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [10- (trimethoxysilyl) undecanamide (polyethylene glycol) -2000] (triethylammonium salt)), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, DSPE-PEG5000 azide (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ azido (polyethylene glycol) -5000] (ammonium salt)), DSPE-PEG2000-square (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ square (polyethylene glycol) -2000] (sodium salt)), DSPE-PEG2000-DBCO (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ dibenzocyclooctyl (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG2000 azide (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ azido (polyethylene glycol) -2000] (ammonium salt)) ], salts, and pharmaceutically acceptable salts thereof, DSPE-PEG2000 biotin (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ biotinyl (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG2000 amine (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ amino (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG2000PDP (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ PDP (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG2000 maleimide (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] (ammonium salt)), (ammonium salt), DSPE-PEG2000 folic acid (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ folate (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG5000 folic acid (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ folate (polyethylene glycol) -5000] (ammonium salt)), DSPE-PEG2000 cyanuric acid (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ cyanuric acid (polyethylene glycol) -2000] (ammonium salt)), DSPE-PEG2000 succinyl group (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ succinyl (polyethylene glycol) -2000] (ammonium salt)), (salts), DSPE-PEG 2000-N-cyanine 5, DSPE-PEG 2000-N-cyanine 7, bis-DSPE-PEG 2000(1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [ azido (polyethylene glycol) -2000 (ammonium salt), DMPE-PEG2000(N- (methylpolyoxyethyleneoxycarbonyl) -1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 2000) ammonium salt)), DMPE-PEG5000(N- (methylpolyoxyethyleneoxycarbonyl) -1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 5,000) ammonium salt)), (ii), DPPE-PEG2000(N- (methylpolyoxyethyleneoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 2000) ammonium salt)), DPPE-PEG5000(N- (methylpolyoxyethyleneoxycarbonyl) -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 5000) ammonium salt)), methoxy-PEG lipids such as 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (ammonium salt), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (ammonium salt), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (ammonium salt), 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (ammonium salt), wherein the methoxy-PEG is 350, 550, 750, 1000, 2000, 3000 or 5000 daltons in weight; or any combination thereof.

In some embodiments, the phospholipid solutions of the present disclosure that can be used to assemble drug-loaded lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) comprise natural phospholipid derivatives, such as Phosphatidylcholine (PC), Phosphatidylglycerol (PG), soy PC, hydrogenated soy PC, or sphingomyelin. In some embodiments, the phospholipid solution used to assemble the lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) loaded with a drug of the present disclosure is a synthetic phospholipid derivative, such as phosphatidic acid (e.g., 1, 2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1, 2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1, 2-distearoyl-sn-glycero-3-phosphate (DSPA)), phosphatidylcholine (e.g., 1, 2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), or a synthetic phospholipid derivative, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dicapryoyl-sn-glycero-3-phosphocholine (DEPC)), phosphatidylglycerols (e.g., 1, 2-dimyristoyl-sn-glycero-3-phospho-rac- (1-glycerol) (DMPG), 1, 2-dipalmitoyl-sn-glycero-3-phospho-rac- (1-glycerol) (DPPG) 1, 2-distearoyl-sn-glycero-3-phospho-rac- (1-glycerol) (DSPG), 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac- (1-glycerol) (POPG)), phosphatidylethanolamines (e.g., 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), and mixtures thereof, Phosphatidylserines (e.g., 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dipalmitoylphosphatidylcholine (DPPtdCho), 1-stearoyl-2- [ (E) -4- (4- ((4-butylphenyl) diazenyl) phenyl) butyryl ] -sn-glycero-3-phosphocholine, 1-stearoyl-2- [ (E) -4- (4- ((4-butylphenyl) diazenyl) phenyl) butyryl ] -sn-glycerol, 1-stearoyl-2- [ (E) -4- (4- ((4-butylphenyl) diazenyl) Phenyl) butyryl ] -sn-glycero-3-phosphocholine, N- [ (E) -4- (4- ((4-butylphenyl) diazenyl) phenyl ] butyryl ] -D-erythro-sphingosylphosphorylcholine, (E) -4- (4- ((4-butylphenyl) diazenyl) phenyl) -N- (3-hydroxy-4-methoxybenzyl) butyramide, 4-butyl-azo-4: 0-oic acid-1, 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt), Bis (1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine) -N ' -diethylenetriaminepentaacetic acid (gadolinium salt), bis (1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) -N ' -diethylenetriaminepentaacetic acid (gadolinium salt), bis (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine) -N ' -diethylenetriaminepentaacetic acid (gadolinium salt), polyglycerol-phospholipid, functionalized phospholipid or a terminally activated phospholipid or a pharmaceutically acceptable salt thereof. In some embodiments, the phospholipid solutions of the present disclosure that can be used to assemble drug-loaded lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) comprise combinations of any of the above phospholipids.

Characterization of lipid-based Carrier formulations

Size: in some embodiments, the lipid-based carriers of the present disclosure (e.g., liposomes, nanodroplets, or microbubbles) can have a diameter of about 70nm to about 10 μm. In some embodiments, the lipid-based carrier of the present disclosure may have about 70nm to about 100nm, about 70nm to about 150nm, about 70nm to about 200nm, about 70nm to about 250nm, about 70nm to about 300nm, about 70nm to about 350nm, about 70nm to about 400nm, about 70nm to about 450nm, about 70nm to about 500nm, about 70nm to about 550nm, about 70nm to about 600nm, about 70nm to about 900nm, about 70nm to about 1 μm, about 70nm to about 5 μm, about 70nm to about 10 μm, about 100nm to about 150nm, about 100nm to about 200nm, about 100nm to about 250nm, about 100nm to about 300nm, about 100nm to about 350nm, about 100nm to about 400nm, about 100nm to about 450nm, about 100nm to about 500nm, about 100nm to about 550nm, about 100nm to about 600nm, about 100nm to about 100 μm, about 100 μm to about 10 μm, About 150nm to about 200nm, about 150nm to about 250nm, about 150nm to about 300nm, about 150nm to about 350nm, about 150nm to about 400nm, about 150nm to about 450nm, about 150nm to about 500nm, about 150nm to about 550nm, about 150nm to about 600nm, about 150nm to about 900nm, about 150nm to about 1 μm, about 150nm to about 5 μm, about 150nm to about 10 μm, about 200nm to about 250nm, about 200nm to about 300nm, about 200nm to about 350nm, about 200nm to about 400nm, about 200nm to about 450nm, about 200nm to about 500nm, about 200nm to about 550nm, about 200nm to about 600nm, about 200nm to about 900nm, about 200nm to about 1 μm, about 200nm to about 5 μm, about 200 to about 10 μm, about 250nm to about 300nm, about 250nm to about 250nm, about 250 to about 250nm, About 250nm to about 900nm, about 250nm to about 1 μm, about 250nm to about 5 μm, about 250nm to about 10 μm, about 300nm to about 350nm, about 300nm to about 400nm, about 300nm to about 450nm, about 300nm to about 500nm, about 300nm to about 550nm, about 300nm to about 600nm, about 300nm to about 900nm, about 300nm to about 1 μm, about 300nm to about 5 μm, about 300nm to about 10 μm, about 350nm to about 400nm, about 350nm to about 450nm, about 350nm to about 500nm, about 350nm to about 550nm, about 350nm to about 600nm, about 350nm to about 900nm, about 350nm to about 1 μm, about 350nm to about 5 μm, about 350nm to about 10 μm, about 400nm to about 450nm, about 400nm to about 500nm, about 400nm to about 550nm, about 400nm to about 1 μm, about 400nm to about 5 μm, about 400nm to about 400nm, about 400 μm to about 400nm to about 400 μm, about 400nm to about 400 μm, About 450nm to about 500nm, about 450nm to about 550nm, about 450nm to about 600nm, about 450nm to about 900nm, about 450nm to about 1 μm, about 450nm to about 5 μm, about 450nm to about 10 μm, about 500nm to about 550nm, about 500nm to about 600nm, about 500nm to about 900nm, about 500nm to about 1 μm, about 500nm to about 5 μm, about 500nm to about 10 μm, about 550nm to about 600nm, about 550nm to about 900nm, about 550nm to about 1 μm, about 550nm to about 5 μm, about 550nm to about 10 μm, about 600nm to about 900nm, about 600nm to about 1 μm, about 600nm to about 5 μm, about 600nm to about 10 μm, about 900nm to about 1 μm, about 900nm to about 5 μm, about 900nm to about 900 μm, about 10 nm to about 10 μm, about 1 μm to about 10 μm, about 10 nm to about 10 μm, or about 10 μm. In some embodiments, the lipid-based carrier of the present disclosure may have a diameter of about 70nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, about 5 μm, or about 10 μm. In some embodiments, the lipid-based carrier of the present disclosure may have a diameter of at least about 70nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, or about 5 μm. In some embodiments, the lipid-based carrier of the present disclosure may have a diameter of up to about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, about 5 μm, or about 10 μm.

The lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can be part of a formulation. In some embodiments, formulations of the present disclosure may comprise a lipid-based carrier having an average particle size of about 70nm to about 10 μm. In some embodiments, the formulations of the present disclosure may comprise a lipid-based carrier having an average particle size of about 70nm to about 100nm, about 70nm to about 150nm, about 70nm to about 200nm, about 70nm to about 250nm, about 70nm to about 300nm, about 70nm to about 350nm, about 70nm to about 400nm, about 70nm to about 450nm, about 70nm to about 500nm, about 70nm to about 550nm, about 70nm to about 600nm, about 70nm to about 900nm, about 70nm to about 1 μm, about 70nm to about 5 μm, about 70nm to about 10 μm, about 100nm to about 150nm, about 100nm to about 200nm, about 100nm to about 250nm, about 100nm to about 300nm, about 100nm to about 350nm, about 100nm to about 400nm, about 100nm to about 450nm, about 100nm to about 500nm, about 100nm to about 550nm, about 100nm to about 600nm, about 1 μm to about 100nm, about 100nm to about 5 μm, About 100nm to about 10 μm, about 150nm to about 200nm, about 150nm to about 250nm, about 150nm to about 300nm, about 150nm to about 350nm, about 150nm to about 400nm, about 150nm to about 450nm, about 150nm to about 500nm, about 150nm to about 550nm, about 150nm to about 600nm, about 150nm to about 900nm, about 150nm to about 1 μm, about 150nm to about 5 μm, about 150nm to about 10 μm, about 200nm to about 250nm, about 200nm to about 300nm, about 200nm to about 350nm, about 200nm to about 400nm, about 200nm to about 450nm, about 200nm to about 500nm, about 200nm to about 550nm, about 200nm to about 600nm, about 200nm to about 900nm, about 200nm to about 1 μm, about 200nm to about 5 μm, about 200 to about 10 μm, about 150nm to about 200nm, about 250nm to about 250nm, about 200nm to about 250nm, About 250nm to about 600nm, about 250nm to about 900nm, about 250nm to about 1 μm, about 250nm to about 5 μm, about 250nm to about 10 μm, about 300nm to about 350nm, about 300nm to about 400nm, about 300nm to about 450nm, about 300nm to about 500nm, about 300nm to about 550nm, about 300nm to about 600nm, about 300nm to about 900nm, about 300nm to about 1 μm, about 300nm to about 5 μm, about 300nm to about 10 μm, about 350nm to about 400nm, about 350nm to about 450nm, about 350nm to about 500nm, about 350nm to about 550nm, about 350nm to about 600nm, about 350nm to about 900nm, about 350nm to about 1 μm, about 350nm to about 5 μm, about 350nm to about 10 μm, about 400nm to about 450nm, about 400nm to about 500nm, about 400nm to about 400 μm, about 400nm to about 1 μm, about 400nm to about 400nm, about 400nm to about 400 μm, About 400nm to about 10 μm, about 450nm to about 500nm, about 450nm to about 550nm, about 450nm to about 600nm, about 450nm to about 900nm, about 450nm to about 1 μm, about 450nm to about 5 μm, about 450nm to about 10 μm, about 500nm to about 550nm, about 500nm to about 600nm, about 500nm to about 900nm, about 500nm to about 1 μm, about 500nm to about 5 μm, about 500nm to about 10 μm, about 550nm to about 600nm, about 550nm to about 900nm, about 550nm to about 1 μm, about 550nm to about 5 μm, about 550nm to about 10 μm, about 600nm to about 900 μm, about 600nm to about 1 μm, about 600nm to about 5 μm, about 600nm to about 10 μm, about 900nm to about 1 μm, about 900nm to about 5 μm, about 10 nm to about 10 μm, about 10 μm to about 10 μm, or about 10 μm to about 10 μm. In some embodiments, the formulations of the present disclosure may comprise a lipid-based carrier having an average particle size of about 70nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, about 5 μm, or about 10 μm. In some embodiments, the formulations of the present disclosure may comprise a lipid-based carrier having an average particle size of at least about 70nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, or about 5 μm. In some embodiments, the formulations of the present disclosure may comprise a lipid-based carrier having an average particle size of up to about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 900nm, about 1 μm, about 5 μm, or about 10 μm.

Load capacity: the loading capacity of a lipid-based carrier (e.g., a liposome, a nanodrop, or a microbubble) is the amount of therapeutic agent (e.g., prodrug) loaded per unit weight of lipid-based carrier. In some embodiments, PLLBCs of the present disclosure have a loading capacity of about 0% to about 99%. In some embodiments, PLLBC of the present disclosure has from about 0% to about 5%, from about 0% to about 10%, from about 0% to about 15%, from about 0% to about 20%, from about 0% to about 30%, from about 0% to about 40%, from about 0% to about 50%, from about 0% to about 75%, from about 0% to about 90%, from about 0% to about 95%, from about 0% to about 99%, from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 30%, from about 5% to about 40%, from about 5% to about 50%, from about 5% to about 75%, from about 5% to about 90%, from about 5% to about 95%, from about 5% to about 99%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 75%, from about 10% to about 90%, from about 10% to about 95%, from about 10% to about 99%, from about 10% to about 20%, from about 20% to about 20%, from about 10% to about 50%, from about 10, About 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 90%, about 15% to about 95%, about 15% to about 99%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 90%, about 20% to about 95%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 75%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 75%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 95%, about 75% to about 95%, about 95% to about 75% to about 99%, about 95% to about 95%, a loading capacity of about 90% to about 99%, or about 95% to about 99%. In some embodiments, PLLBCs of the present disclosure have a loading capacity of about 0%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, about 95%, or about 99%. In some embodiments, PLLBCs of the present disclosure have a loading capacity of at least about 0%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, or about 95%. In some embodiments, PLLBCs of the present disclosure have a loading capacity of at most about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, about 95%, or about 99%.

The loading capacity of a formulation of lipid-based carriers (e.g., liposomes, nanodroplets, or microbubbles) is the amount of therapeutic agent (e.g., prodrug) per unit weight of lipid-based carrier loaded into the lipid-based carrier of the formulation in the formulation. In some embodiments, the formulations of PLLBCs of the present disclosure have a loading capacity of about 0% to about 99%. In some embodiments, PLLBC of the present disclosure has a formulation of about 0% to about 5%, about 0% to about 10%, about 0% to about 15%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 75%, about 0% to about 90%, about 0% to about 95%, about 0% to about 99%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 90%, about 5% to about 95%, about 5% to about 99%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 90%, about 10% to about 95%, about 10% to about 99%, about 10% to about 50%, about 10%, or a, About 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 75%, about 15% to about 90%, about 15% to about 95%, about 15% to about 99%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 90%, about 20% to about 95%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 75%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 75%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 75% to about 75%, about 75% to about 75%, about 50% to about 95%, about 75% to about 99%, or a, A loading capacity of about 90% to about 95%, about 90% to about 99%, or about 95% to about 99%. In some embodiments, the formulations of PLLBCs of the present disclosure have a loading capacity of about 0%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, about 95%, or about 99%. In some embodiments, the formulations of PLLBCs of the present disclosure have a loading capacity of at least about 0%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, or about 95%. In some embodiments, the formulations of PLLBCs of the present disclosure have a loading capacity of up to about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 75%, about 90%, about 95%, or about 99%.

Prodrug incorporation: prodrugs incorporated into PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can be measured in terms of the moles of prodrug to the total moles of phospholipid (mol%). In some embodiments, the prodrug incorporated into PLLBC of the present disclosure may be from about 1 mol% to about 100 mol%. In some embodiments, a prodrug incorporated into a PLLBC of the present disclosure may be about 1 mol% to about 10 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 30 mol%, about 1 mol% to about 40 mol%, about 1 mol% to about 50 mol%, about 1 mol% to about 60 mol%, about 1 mol% to about 70 mol%, about 1 mol% to about 80 mol%, about 1 mol% to about 90 mol%, about 1 mol% to about 100 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 40 mol%, about 10 mol% to about 50 mol%, about 10 mol% to about 60 mol%, about 10 mol% to about 70 mol%, about 10 mol% to about 80 mol%, about 10 mol% to about 90 mol%, about 10 mol% to about 100 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 40 mol%, about 20 mol% to about 20 mol%, about 20 mol% to about 50 mol%, about 20 mol% to about 20 mol%, about 20 mol% to about 70 mol%, or a combination thereof, About 20 mol% to about 80 mol%, about 20 mol% to about 90 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 80 mol%, about 30 mol% to about 90 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 60 mol%, about 40 mol% to about 70 mol%, about 40 mol% to about 80 mol%, about 40 mol% to about 90 mol%, about 40 mol% to about 100 mol%, about 50 mol% to about 60 mol%, about 50 mol% to about 70 mol%, about 50 mol% to about 80 mol%, about 50 mol% to about 100 mol%, about 60 mol% to about 70 mol%, about 60 mol% to about 80 mol%, about 60 mol% to about 60 mol%, about 60 mol% to about 90 mol%, about 60 mol% to about 60 mol%, about 60 mol% to about 80 mol%, about 80 mol% to about 70 mol%, about 30 mol% to about 80 mol%, about 70 mol% to about 90 mol%, about 70 mol% to about 100 mol%, about 80 mol% to about 90 mol%, about 80 mol% to about 100 mol%, or about 90 mol% to about 100 mol%. In some embodiments, the prodrug incorporated into PLLBC of the present disclosure may be about 1 mol%, about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, about 70 mol%, about 80 mol%, about 90 mol%, or about 100 mol%. In some embodiments, the prodrug incorporated into PLLBC of the present disclosure may be at least about 1 mol%, about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, about 70 mol%, about 80 mol%, or about 90 mol%. In some embodiments, the prodrug incorporated into PLLBC of the present disclosure may be up to about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, about 70 mol%, about 80 mol%, about 90 mol%, or about 100 mol%.

Stability: the ability of PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) to maintain a constant size is a measure of stability. In some embodiments, PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure are stable for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or about 5 years.

Synthesis and self-assembly

Fig. 2 presents a non-limiting example of PLLBC. Figure 2 panel a shows the synthesis of prodrugs, self-assembly of prodrug-loaded liposomes and the use of liposomes for treating cells in vitro. Figure 2 panel B shows the use of prodrug-loaded microbubbles and ultrasound exposure for targeted drug delivery in vitro.

Mode of administration

PLLBCs of the present disclosure, such as liposomes and microbubbles, can be administered in single or multiple doses to treat a condition. Administration of PLLBCs of the present disclosure can be carried out in various forms and routes, including, for example, intravenous, intraarterial, subcutaneous, intramuscular, oral, parenteral, ocular, subcutaneous, transdermal, nasal, vaginal, and topical administration. In some embodiments, PLLBCs of the present disclosure may be administered locally, e.g., by direct injection into an organ.

Application amount and frequency

Formulations of PLLBCs of the present disclosure, such as liposomes and microbubbles, can be prepared in unit dosage forms suitable for single administration of precise dosages. In unit dosage forms, formulations of PLLBCs of the present disclosure are divided into unit doses containing appropriate amounts of the prodrug. The unit dose can be in the form of a package containing discrete quantities of the formulation. A non-limiting example is a liquid in a vial or ampoule. The aqueous suspension composition may be packaged in a non-reclosable single dose container. The reclosable multi-dose container can be used in combination with, for example, a preservative. Formulations for parenteral injection may be presented in unit dosage form in, for example, ampoules or in multi-dose containers with a preservative.

PLLBC of the present disclosure may be present in the formulation in a unit dosage form of about 0mg/mL to about 400 mg/mL. The PLLBC of the present disclosure may be administered in a range of about 0mg/mL to about 5mg/mL, about 0mg/mL to about 10mg/mL, about 0mg/mL to about 15mg/mL, about 0mg/mL to about 20mg/mL, about 0mg/mL to about 25mg/mL, about 0mg/mL to about 50mg/mL, about 0mg/mL to about 75mg/mL, about 0mg/mL to about 100mg/mL, about 0mg/mL to about 200mg/mL, about 0mg/mL to about 300mg/mL, about 0mg/mL to about 400mg/mL, about 5mg/mL to about 10mg/mL, about 5mg/mL to about 15mg/mL, about 5mg/mL to about 20mg/mL, about 5mg/mL to about 25mg/mL, about 5mg/mL to about 50mg/mL, about 50mg/mL, About 5mg/mL to about 75mg/mL, about 5mg/mL to about 100mg/mL, about 5mg/mL to about 200mg/mL, about 5mg/mL to about 300mg/mL, about 5mg/mL to about 400mg/mL, about 10mg/mL to about 15mg/mL, about 10mg/mL to about 20mg/mL, about 10mg/mL to about 25mg/mL, about 10mg/mL to about 50mg/mL, about 10mg/mL to about 75mg/mL, about 10mg/mL to about 100mg/mL, about 10mg/mL to about 200mg/mL, about 10mg/mL to about 300mg/mL, about 10mg/mL to about 400mg/mL, about 15mg/mL to about 20mg/mL, about 15mg/mL to about 25mg/mL, about 15mg/mL to about 50mg/mL, or, About 15mg/mL to about 75mg/mL, about 15mg/mL to about 100mg/mL, about 15mg/mL to about 200mg/mL, about 15mg/mL to about 300mg/mL, about 15mg/mL to about 400mg/mL, about 20mg/mL to about 25mg/mL, about 20mg/mL to about 50mg/mL, about 20mg/mL to about 75mg/mL, about 20mg/mL to about 100mg/mL, about 20mg/mL to about 200mg/mL, about 20mg/mL to about 300mg/mL, about 20mg/mL to about 400mg/mL, about 25mg/mL to about 50mg/mL, about 25mg/mL to about 75mg/mL, about 25mg/mL to about 100mg/mL, about 25mg/mL to about 200mg/mL, about 25mg/mL to about 300mg/mL, about, About 25mg/mL to about 400mg/mL, about 50mg/mL to about 75mg/mL, about 50mg/mL to about 100mg/mL, about 50mg/mL to about 200mg/mL, about 50mg/mL to about 300mg/mL, about 50mg/mL to about 400mg/mL, about 75mg/mL to about 100mg/mL, about 75mg/mL to about 200mg/mL, about 75mg/mL to about 300mg/mL, about 75mg/mL to about 400mg/mL, about 100mg/mL to about 200mg/mL, about 100mg/mL to about 300mg/mL, about 100mg/mL to about 400mg/mL, about 200mg/mL to about 300mg/mL, about 200mg/mL to about 400mg/mL, or from about 300mg/mL to about 400mg/mL of the unit dosage form is present in the formulation. PLLBCs of the present disclosure can be present in a formulation in a unit dosage form of about 0mg/mL, about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 50mg/mL, about 75mg/mL, about 100mg/mL, about 200mg/mL, about 300mg/mL, or about 400 mg/mL. PLLBCs of the present disclosure can be present in a formulation in a unit dosage form of at least about 0mg/mL, about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 50mg/mL, about 75mg/mL, about 100mg/mL, about 200mg/mL, or about 300 mg/mL. PLLBCs of the present disclosure may be present in a formulation in a unit dosage form of up to about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 50mg/mL, about 75mg/mL, about 100mg/mL, about 200mg/mL, about 300mg/mL, or about 400 mg/mL.

The dosage level of PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure may depend on a variety of factors including, for example, the activity of the PLLBC used, the route of administration, the time of administration, the rate of excretion, the metabolism of the prodrug, the duration of treatment, the prodrug compound used in conjunction with the PLLBC, the compound and/or material, the age, sex, weight, condition, general health, and past medical history of the subject being treated. Dosage values may also vary with the severity of the condition being treated. For any particular subject, the particular dosage regimen can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition.

In some embodiments, the dose may be expressed as the amount of prodrug divided by the mass of the subject, e.g., milligrams of prodrug per kilogram of body weight of the subject. In some embodiments, the dose is administered in an amount of about 5mg/kg to about 50mg/kg, 250mg/kg to about 2000mg/kg, about 10mg/kg to about 800mg/kg, about 50mg/kg to about 400mg/kg, about 100mg/kg to about 300mg/kg, or about 150mg/kg to about 200 mg/kg.

In some embodiments, the dose can be expressed in terms of the amount of PLLBC (e.g., liposomes, nanodroplets, or microbubbles) per kilogram of the subject's body weight. In some embodiments, the dose is administered in an amount of about 5mg/kg to about 50mg/kg, 250mg/kg to about 2000mg/kg, about 10mg/kg to about 800mg/kg, about 50mg/kg to about 400mg/kg, about 100mg/kg to about 300mg/kg, or about 150mg/kg to about 200 mg/kg.

Combination therapy

In some embodiments, PLLBCs of the present disclosure include a plurality of prodrugs. Administration of PLLBC comprising a combination of prodrugs may have a synergistic effect. Synergy may refer to the observation that the overall effect resulting from administration of PLLBC comprising multiple prodrugs may be greater than the sum of the individual effects of administration of PLLBC comprising one prodrug. Synergistic effect may also refer to the observation that PLLBC with one prodrug produces little or no effect, but PLLBC with multiple prodrugs produces a greater effect than PLLBC with only a second prodrug. Synergistic effect may also refer to the observation that administration of PLLBC with multiple prodrugs to a subject reduces side effects in the subject compared to administration of PLLBC with one prodrug

In some embodiments, lipid-based carriers loaded with different prodrugs can be administered to a subject in combination. The combined administration of PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) can have a synergistic effect. Synergy may refer to the observation that the combination of two prodrug-loaded lipid-based carriers may produce an overall effect that is greater than the sum of the two individual effects. Synergistic may also mean that a single PLLBC produces little or no effect, but when administered with a second PLLBC, produces an effect greater than that produced by the second PLLBC alone. Synergistic effect may also refer to the observation that administration of two PLLBCs in combination to a subject reduces side effects in the subject compared to administration of PLLBCs alone. Administration of different PLLBCs can be carried out simultaneously or sequentially by the same or different routes of administration.

Triggering prodrug activity at target sites

In some embodiments, PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can deliver the prodrug to a target site in a subject. The target site may be, for example, a site of a local infection, cancerous lesion, non-cancerous lesion, metastatic lesion, pre-cancerous lesion, tumor, organ, or a specific cell type, such as red blood cells, white blood cells, neutrophils, macrophages, or neurons. Delivery of the prodrug to the target site can, for example, reduce the dosage required to effectively treat the disorder, increase the effectiveness of the prodrug, or reduce side effects caused by the prodrug in the subject. In some embodiments, a prodrug present at the target site as part of PLLBC may remain inactive in the absence of an in vitro trigger. PLLBC can be triggered once an in vitro trigger is present, resulting in the release and activation of the prodrug. Non-limiting examples of in vitro triggers include ultrasound, magnetic fields, electric fields, light waves, and radiation. A schematic of a microbubble therapy using ultrasound as an in vitro trigger is shown in figure 3.

Ultrasound is a sound wave with a frequency above the highest audible upper limit of human hearing. In some embodiments, PLLBC of the present disclosure (e.g., liposomes, microbubbles, or nanodroplets) can be triggered using an ultrasound frequency of about 1MHz to about 20 MHz. In some embodiments, about 1MHz to about 2MHz, about 1MHz to about 3MHz, about 1MHz to about 4MHz, about 1MHz to about 5MHz, about 1MHz to about 6MHz, about 1MHz to about 7MHz, about 1MHz to about 8MHz, about 1MHz to about 9MHz, about 1MHz to about 10MHz, about 1MHz to about 15MHz, about 1MHz to about 20MHz, about 2MHz to about 3MHz, about 2MHz to about 4MHz, about 2MHz to about 5MHz, about 2MHz to about 6MHz, about 2MHz to about 7MHz, about 2MHz to about 8MHz, about 2MHz to about 9MHz, about 2MHz to about 10MHz, about 2MHz to about 15MHz, about 2MHz to about 20MHz, about 3MHz to about 4MHz, about 3MHz to about 5MHz, about 3MHz to about 6MHz, about 3MHz to about 7MHz, about 3MHz to about 8MHz, about 3MHz to about 9MHz, about 3MHz to about 3MHz, about 3MHz to about 4MHz, about 3MHz to about 5MHz, about 3MHz to about 3MHz, about 3MHz to about 5MHz, about 3MHz, about 10MHz, about 3MHz to about 3MHz, about 5MHz, about 3MHz to about 6MHz, about 6MHz, About 4MHz to about 6MHz about 4MHz to about 7MHz, about 4MHz to about 8MHz, about 4MHz to about 9MHz, about 4MHz to about 10MHz, about 4MHz to about 15MHz, about 4MHz to about 20MHz, about 5MHz to about 6MHz, about 5MHz to about 7MHz, about 5MHz to about 8MHz, about 5MHz to about 9MHz, about 5MHz to about 10MHz, about 5MHz to about 15MHz, about 5MHz to about 20MHz, about 6MHz to about 7MHz, about 6MHz to about 8MHz, about 6MHz to about 9MHz, about 6MHz to about 10MHz, about 6MHz to about 15MHz, about 6MHz to about 20MHz, about 7MHz to about 8MHz, about 7MHz to about 9MHz, about 7MHz to about 10MHz, about 7MHz to about 15MHz, about 7MHz to about 20MHz, about 8MHz to about 9MHz, about 8MHz to about 10MHz, about 8MHz to about 15MHz, about 9MHz to about 9MHz, about 7MHz to about 10MHz, about 9MHz to about 9MHz, about 10MHz, about 9MHz, An ultrasonic frequency of about 10MHz to about 20MHz or about 15MHz to about 20MHz triggers PLLBC of the present disclosure. In some embodiments, an ultrasound frequency of about 1MHz, about 2MHz, about 3MHz, about 4MHz, about 5MHz, about 6MHz, about 7MHz, about 8MHz, about 9MHz, about 10MHz, about 15MHz, or about 20MHz may be used to trigger PLLBC of the present disclosure. In some embodiments, an ultrasound frequency of at least about 1MHz, about 2MHz, about 3MHz, about 4MHz, about 5MHz, about 6MHz, about 7MHz, about 8MHz, about 9MHz, about 10MHz, or about 15MHz may be used to trigger PLLBC of the present disclosure. In some embodiments, ultrasound frequencies up to about 2MHz, about 3MHz, about 4MHz, about 5MHz, about 6MHz, about 7MHz, about 8MHz, about 9MHz, about 10MHz, about 15MHz, or about 20MHz may be used to trigger PLLBC of the present disclosure.

In some embodiments, the ultrasound may generate pressure on the PLLBC. In some embodiments, the pressure is a pressure of about 25kPa to about 2.5 MPa. In some embodiments, the pressure is from about 25kPa to about 300 kPa. In some embodiments, the pressure is a pressure of about 1MPa to about 2.5 MPa.

In some embodiments, light may be used to trigger PLLBC, such as liposomes, nanodroplets, or microbubbles. In some embodiments, the light is in the form of laser pulses. In some embodiments, the wavelength of the light may be from about 400nm to about 1,400 nm. In some embodiments, the wavelength of light may be from about 400nm to about 450nm, from about 400nm to about 500nm, from about 400nm to about 550nm, from about 400nm to about 600nm, from about 400nm to about 650nm, from about 400nm to about 700nm, from about 400nm to about 750nm, from about 400nm to about 800nm, from about 400nm to about 900nm, from about 400nm to about 1,000nm, from about 400nm to about 1,400nm, from about 450nm to about 500nm, from about 450nm to about 550nm, from about 450nm to about 600nm, from about 450nm to about 650nm, from about 450nm to about 700nm, from about 450nm to about 750nm, from about 450nm to about 800nm, from about 450nm to about 900nm, from about 450nm to about 1,000nm, from about 450nm to about 1,400nm, from about 500nm to about 550nm, from about 500nm to about 600nm, from about 500nm to about 500nm, about 500nm, About 550nm to about 650nm, about 550nm to about 700nm, about 550nm to about 750nm, about 550nm to about 800nm, about 550nm to about 900nm, about 550nm to about 1,000nm, about 550nm to about 1,400nm, about 600nm to about 650nm, about 600nm to about 700nm, about 600nm to about 750nm, about 600nm to about 800nm, about 600nm to about 900nm, about 600nm to about 1,000nm, about 600nm to about 1,400nm, about 650nm to about 700nm, about 650nm to about 750nm, about 650nm to about 800nm, about 650nm to about 900nm, about 650nm to about 1,000nm, about 650nm to about 1,400nm, about 700nm to about 750nm, about 700nm to about 800nm, about 700nm to about 900nm, about 700nm to about 1,000nm, about 700nm to about 1,400nm, about 1nm to about 750nm, about 1,000nm, about 1,400nm to about 750nm, about 1,000nm to about 750nm, about 1,000nm, about 700nm to about 900nm, about 800nm to about 1,000nm, about 800nm to about 800nm, about 1,000nm, about 800nm to about, From about 900nm to about 1,400nm or from about 1,000nm to about 1,400 nm. In some embodiments, the wavelength of light may be about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 650nm, about 700nm, about 750nm, about 800nm, about 900nm, about 1,000nm, or about 1,400 nm. In some embodiments, the wavelength of light may be at least about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 650nm, about 700nm, about 750nm, about 800nm, about 900nm, or about 1,000 nm. In some embodiments, the wavelength of light may be up to about 450nm, about 500nm, about 550nm, about 600nm, about 650nm, about 700nm, about 750nm, about 800nm, about 900nm, about 1,000nm, or about 1,400 nm.

In some embodiments, a magnetic field may be used to trigger PLLBC, such as liposomes, nanodroplets, or microbubbles. In some embodiments, the magnetic field strength is from 0.2T to about 7T. In some embodiments, the magnetic field strength is about 0.2T to about 0.5T, about 0.2T to about 1T, about 0.2T to about 1.5T, about 0.2T to about 2T, about 0.2T to about 3T, about 0.2T to about 4T, about 0.2T to about 5T, about 0.2T to about 6T, about 0.2T to about 7T, about 0.5T to about 1T, about 0.5T to about 1.5T, about 0.5T to about 2T, about 0.5T to about 3T, about 0.5T to about 4T, about 0.5T to about 5T, about 0.5T to about 6T, about 0.5T to about 7T, about 1T to about 1.5T, about 1T to about 2T, about 1T to about 3T, about 1T to about 4T, about 1T to about 5T, about 1T to about 1.5T, about 1T to about 1T, about 1T to about 2T, about 1T, about 5T, about 1T to about 5T, about 1T, about 5T, about 1T to about 5T, about 1T, about 5T, about 1T, about 5T, about 2T, about 2T to about 4T, about 2T to about 5T, about 2T to about 6T, about 2T to about 7T, about 3T to about 4T, about 3T to about 5T, about 3T to about 6T, about 3T to about 7T, about 4T to about 5T, about 4T to about 6T, about 4T to about 7T, about 5T to about 6T, about 5T to about 7T, or about 6T to about 7T. In some embodiments, the magnetic field strength is about 0.2T, about 0.5T, about 1T, about 1.5T, about 2T, about 3T, about 4T, about 5T, about 6T, or about 7T. In some embodiments, the magnetic field strength is at least about 0.2T, about 0.5T, about 1T, about 1.5T, about 2T, about 3T, about 4T, about 5T, or about 6T. In some embodiments, the magnetic field strength is at most about 0.5T, about 1T, about 1.5T, about 2T, about 3T, about 4T, about 5T, about 6T, or about 7T.

The amount of time the in vitro trigger is applied to the subject can vary. In some embodiments, the in vitro trigger is applied for about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or about 12 hours. In some embodiments, the in vitro trigger is applied for about 1 second to about 30 seconds, about 1 minute to about 30 minutes, or about 1 hour to about 6 hours, or about 6 hours to about 12 hours.

In some embodiments, the in vitro trigger is applied to the subject in the form of a pulse. In some embodiments, the in vitro trigger is applied in 2-100 pulses. In some embodiments, the in vitro trigger may be applied in 2,3, 4,5, 6,7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pulses. In some embodiments, the in vitro trigger is applied in more than 100 pulses. In some embodiments, each pulse of the in vitro trigger is applied for about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or about 12 hours. In some embodiments, the pulse of in vitro trigger is applied for about 1 second to about 30 seconds, about 1 minute to about 30 minutes, or about 1 hour to about 6 hours, or about 6 hours to about 12 hours.

Contrast agents

In some embodiments, PLLBCs of the present disclosure, such as liposomes, nanodroplets, or microbubbles, can act as contrast agents. Contrast agents may improve imaging techniques, such as ultrasound imaging. Ultrasound imaging is portable, can provide real-time imaging feedback, and does not risk ionizing radiation. Ultrasound contrast agents (e.g., microbubbles) produce a nonlinear response to ultrasound and provide a high signal-to-noise ratio for cardiology imaging. The ability of ultrasound to visualize a target tissue region with microbubbles in real time may allow for measurements of, for example, tumor size, vasculature, and blood flow.

Treatment of disorders

PLLBCs of the present disclosure, such as liposomes, nanodroplets, or microbubbles, can be used to treat, prevent, or diagnose disorders. In some embodiments, PLLBCs of the present disclosure can be used to treat, prevent, or diagnose a disorder, such as cancer, a viral infection, a bacterial infection, an inflammatory disorder, or a neurological disorder.

PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can be used to treat, prevent, or diagnose cancer. In some embodiments, PLLBCs of the present disclosure can be used to treat cancer, for example, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma, primary central nervous system lymphoma, anal carcinoma, gastrointestinal carcinoid tumors, astrocytoma, atypical teratoid/rhabdoid tumors, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain tumor, breast carcinoma, bronchial tumor, burkitt's lymphoma, non-hodgkin's lymphoma, carcinoid tumors, cardiac tumors, embryonic tumors, germ cell tumors, cervical carcinoma, cholangiocarcinoma, chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative tumor, colorectal carcinoma, craniopharyngeal tumor, cutaneous T-cell lymphoma, Ductal Carcinoma In Situ (DCIS), Endometrial (uterine) cancer, ependymoma, esophageal cancer, olfactory neuroblastoma (head and neck cancer), ewing's sarcoma, extracranial germ cell tumor, intraocular melanoma, retinoblastoma, fallopian tube cancer, bone fibroblastic tumor, osteosarcoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, hypopharynx cancer, intraocular melanoma, islet cell tumor of pancreas, pancreatic neuroendocrine tumor, renal (kidney) cancer, langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, non-small cell lung cancer, lymphoma, malignant fibrous histiocytoma of bone, osteosarcoma, melanoma, neuroblastoma, merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer, midline cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides (lymphoma), nasal and sinus cancer, neuroblastoma, non-Hodgkin lymphoma, pancreatic cancer, pancreatic neuroendocrine tumor, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleural pneumocyte tumor, primary peritoneal, prostate, rectal, rhabdomyosarcoma, salivary gland, vascular tumors, Sezary syndrome (lymphoma), small bowel, soft tissue sarcoma, T-cell lymphoma, testicular, throat, nasopharyngeal, oropharyngeal, hypopharyngeal, thymus and thymus cancers, thyroid, urinary tract, uterine sarcoma, vaginal, vascular, vulval, or wilms tumors.

PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can be used to treat, prevent, or diagnose viral infections. In some embodiments, PLLBCs of the present disclosure can be used to treat respiratory viral infections, such as influenza, respiratory syncytial virus infection, adenovirus infection, parainfluenza virus infection, or Severe Acute Respiratory Syndrome (SARS). In some embodiments, PLLBCs of the present disclosure can be used to treat gastrointestinal viral infections, such as norovirus infection, rotavirus infection, adenovirus infection, or astrovirus infection. In some embodiments, PLLBCs of the present disclosure can be used to treat a eruptive viral infection, such as measles, rubella, varicella, shingles, rubella, smallpox, fifth disease, or chikungunya viral infection. In some embodiments, the liposomes, nanodroplets, or microvesicles of the present disclosure may be used to treat a liver viral infection, such as hepatitis a, hepatitis b, hepatitis c, hepatitis d, or hepatitis e. In some embodiments, PLLBCs of the present disclosure can be used to treat a cutaneous viral infection, such as a wart (e.g., a genital wart), an oral herpes, a genital herpes, or a molluscum contagiosum. In some embodiments, PLLBCs of the present disclosure can be used to treat hemorrhagic viral diseases, such as ebola, lassa fever, dengue fever, yellow fever, marburg hemorrhagic fever, or crimiania-congo hemorrhagic fever. In some embodiments, PLLBCs of the present disclosure can be used to treat a neurological viral infection, such as polio, viral meningitis, viral encephalitis, or rabies.

PLLBCs (e.g., liposomes, nanodroplets, or microbubbles) of the present disclosure can be used to treat, prevent, or diagnose bacterial infections. In some embodiments, PLLBCs of the present disclosure can be used to treat or prevent bacterial infections, such as Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus saprophyticus (Staphylococcus saprophyticus), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus bovis (Streptococcus bovis), Streptococcus pneumoniae (Streptococcus pneoniae), Streptococcus viridans (Streptococcus streptococci), Bacillus anthracis (Bacillus), Bacillus cereus (Bacillus cereus), Clostridium tetani (Clostridium tetani), Clostridium botulinum (Clostridium tetani), Clostridium tetani (Clostridium perfringens), Clostridium perfringens (Clostridium perfringens), Clostridium difficile (Clostridium difficile), Clostridium histolyticum (Clostridium histolyticum), Clostridium histolyticum (Streptococcus pneumoniae), Clostridium histolyticum), Clostridium perfringens (Streptococcus pneumoniae), Clostridium histolyticum (Streptococcus pneumoniae), Clostridium histophilus (Streptococcus pneumoniae), Clostridium histophilus), Clostridium histolyticum (Clostridium histophilus), Clostridium histophilus (Clostridium histophilus), Clostridium histoplasmosis (Clostridium histoplasmosis), Clostridium histoplasmosis (Clostridium histophilus), shigella (Shigella) (bacillary dysentery), Vibrio cholerae (Vibrio cholerae), Campylobacter jejuni (Campylobacter jejuni), or Helicobacter pylori (Helicobacter pylori).

Examples

Example 1: synthesis method

Scheme 1 describes a synthetic route for coupling two chemotherapeutic compounds (denoted as P and N) to a linker comprising two phospholipid chains.

Scheme 1

A mixture of the parent compound (0.24mmol, 1 equiv.), DCC (0.73mmol, 3 equiv.), DPPE-Glu (0.24mmol, 1 equiv.), and DMAP (0.048mmol, 0.4 equiv.) was charged to a 10mL flask. Under a nitrogen atmosphere, 5.5mL of dry THF was added to the flask. The reaction mixture was stirred at room temperature for 24 hours. Thin Layer Chromatography (TLC) plates were used to monitor the reaction and to guide all flash column chromatography (Kiesel gel 60, 230-400 mesh). High resolution mass spectrometry is used to verify the chemical structure of the prodrug. The hydrophobic fatty acid of each prodrug acts as an anchor to be incorporated into the lipid layer of the lipophilic drug delivery vehicle.

2, 3-bis (palmitoyloxy) propyl (2- (5-oxo-5- ((((5S,5aS,8aS,9S) -8-oxo-9- (3,4, 5-trimethoxyphenyl) -5,5a,6,8,8a, 9-hexahydrofuro [3',4':6,7]Naphtho [2,3-d ]][1,3]Dioxol-5-yl) oxy) pentanamido) ethyl) sodium phosphate (2T-P). Yield 28.6%, white solid, mp 60 ℃ (CH)₂Cl₂/MeOH＝7/1)。¹H NMR(CDCl₃–d₆)6.83(s,1H),6.53(s,1H),6.39(s,2H),5.98(s,2H),5.91(d,J＝7.04Hz,1H),5.26(s,1H),4.58(d,J＝3.32,1H),4.37(s,2H),4.23-4.15(m,2H),3.94(s,4H),3.79(d,J＝19.2Hz,9H),3.50(s,2H),2.89-2.82(m,2H),2.56-2.50(m,2H),2.35-2.19(m,6H),2.12-2.01(m,7H),1.31(s,48H),0.91-0.88(m,6H)；¹³C NMR(CDCl₃–d₆)173.8,152.6,148.1,147.5,134.9,132.4,108.2,101.7,60.7,56.2,38.7,34.5,34.3,33.5,31.9,29.8,29.7,29.4,25,24.9,22.7,14.1。

2, 3-bis (palmitoyloxy) propyl (2- (5- ((7- (3, 5-dibromophenyl) -8-oxo-7, 8,10, 11-tetrahydrobenzo [ h)]Furo [3,4-b ]]Quinolin-2-yl) oxy) -5-oxopentanamido) ethyl) sodium phosphate (2T-N). Yield 25.6%, oil, (CH)₂Cl₂/MeOH＝7/1)。¹H NMR(CDCl₃–d₆)10.56(s,1H),8.06(s,1H),7.89(d,J＝6.88Hz,1H)；7.72(d,J＝8.96Hz,1H),7.59(s,1H),7.44(s,1H),7.37-7.29(m,3H),7.19(d,J＝8.8Hz,1H),6.96(d,J＝8.36,1H),6.38(d,J＝7.08Hz,1H),5.25(s,1H),5.13-4.99(m,3H),4.34(d,J＝,1H),4.02-4.01(m,5H),3.51(d,J＝14.08Hz,2H),3.05(s,3H),2.69(s,2H),2.41(s,2H),2.23(d,J＝6Hz,4H),2.09(s,2H),1.31-1.21(m,46H)0.91-0.89(m,6H)；¹³C NMR(CDCl₃–d₆)173.6,173.4,173.2,173.2,172.4 158.9,156.6,149.8,148.8,139.7,132.4,131.9,131.2,130.2,129.6,128.0,123.7,123.1,121.6,118.6,106.4,96.1,66.5,64.5,62.5,40.9,39.8,34.9,34.2,34.0,32.7,31.9,29.7,29.7,29.6,29.6,29.4,29.4,29.2,29.1,24.9,24.8,22.7,20.9,14.1。

Figures 1 and 4 show synthetic routes for coupling topotecan and cytarabine to phospholipids. Phospholipid conjugation via amino attachment (cytarabine) or hydroxyl attachment (cytarabine, topotecan) to yield the two-tailed topotecan (2T-T) or the two-tailed cytarabine (2T-C). The progress of the reaction was checked using thin layer chromatography and by using 3:1CH₂Cl₂:MeOH(2T-C,NH₂)、1:1CH₂Cl₂MeOH (2T-C, OH) and 4:1CHCl₃MeOH (2T-T) to purify the prodrug. By passing¹H and C¹³Nuclear magnetic resonance spectroscopy (NMR) confirmed the structure of the prodrug and found that the yield of 2T-C (amino) was 23%, the yield of 2T-C (OH) was 7%, and the yield of 2T-T was 19%.

Example 2: liposome suspensions

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is used at the desired mol%; 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol) 2000) ammonium salt (DSPE-PEG2000) in chloroform; and prodrug in chloroform to prepare liposome prodrug-loaded lipid membranes. The lipid mixture was then dried under nitrogen and further dried under vacuum at 50 ℃ for 2 hours. The prodrug-rich lipid membrane was resuspended in a 0.5mL aliquot of 1X Phosphate Buffered Saline (PBS) solution using an ultrasonic bath for 30 minutes at 50 ℃ to provide 1mg of lipid per 1mL of PBS liposome suspension.

Example 3: microbubble suspensions

To generate 2T-N loaded microvesicles, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used at the required mol%; 1, 2-dipalmitoyl-sn-glycero-3-phosphate (monosodium salt) (DPPA); 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- (folate (polyethylene glycol) -5000) (ammonium salt) (DSPE-PEG5000) in chloroform; and a solution of the prodrug in chloroform to prepare a lipid membrane loaded with the 2T-N prodrug. The lipid mixture was then dried under nitrogen and further dried under vacuum at 50 ℃ for 2 hours. The prodrug-rich lipid membrane was resuspended in a 1.5mL aliquot of (80 vol% 0.1M Tris, 10 vol% glycerol, 10 vol% propylene glycol) Tris buffer using an ultrasonic bath for 30 minutes at 50 ℃ to give 1.5mg of lipid per 1.5mL of Tris-buffered liposome suspension. After sealing, 10mL of sulfur hexafluoride (SF) was used₆) Purge each vial. The vial was shaken for 45 seconds using a mechanical shaker to form microbubbles from the liposome suspension.

Example 4: nanopallet suspensions

DSPC: DSPE-PEG2000 was formed and used to generate Nanodroplets (ND) with perfluorobutane. Submerging the vial containing the microbubbles in 5 ℃ CO₂In an isopropanol bath and vented with a 27G needle, then pressurized with about 30mL of air (from the room). Freezing of the lipids was prevented by observing the vial contents and bath temperature.

Example 5: differential scanning calorimetry

Prodrug-loaded liposome (PLL) samples with increasing prodrug concentrations were prepared without extrusion using 20mg of lipid per 1mL of PBS for each compound. Deionized water was used as a calibration standard. 10 μ L of each liposome suspension was transferred and sealed in an aluminum DSC pan. The DSC measurement was performed at room temperature, and then the sample was heated from 15 ℃ to 55 ℃ at 5 ℃/min.

Figure 5 shows the differential scanning calorimetry curve of 2T-P loaded liposomes at increasing concentrations. Figure 6 shows the differential scanning calorimetry curve of increasing concentrations of P-loaded liposomes.

DSC is a thermal analysis technique that can be used to design lipid drug delivery systems (e.g., liposomes) by determining the compatibility of mixed molecules and compositions with the liposome bilayer. The thermotropic behavior of liposomes at different prodrug concentrations was measured to determine if insertion of the molecule altered the phase transition temperature. PEG-2000 masks the phase front transition. The thermograms of PLL (2T-P and 2T-N; 0 to 37 mol%) compared to empty liposomes show a gradual decrease of the phase transition temperature with increasing prodrug composition. The thermograms of P-and N-loaded liposomes (0 to 31 mol%) maintained the phase transition temperature with little decrease in the endothermic peak. The reduction in the endothermic peak of 2T-P-and 2T-N loaded liposomes indicates a change in lipid-lipid interactions due to the presence of the prodrug in the bilayer. P at 13 mol% resulted in a slight change in phase transition, corresponding to drug incorporation (fig. 7 panel a and fig. 7 panel B). As the concentration increases, the phase transition returns to that of the empty liposomes. Higher concentrations of P lead to the formation of micelles, rather than entrapping P in the bilayer of the liposome. N does not lower the phase transition temperature. All liposome suspensions used for DSC analysis were prepared in deionized water instead of sodium buffer to prevent unwanted interactions. The sample was not extruded.

Example 6: liposome particle size distribution and stability determination

Liposome size distribution was characterized by Dynamic Light Scattering (DLS). Measurements were performed using disposable polystyrene size cuvettes containing liposome suspensions. The reported DLS measurements are the average of 3 individually prepared liposome samples.

Example 7: 2T-P and 2T-N incorporation efficiency measurements

Measuring the concentration of parent compound and prodrug in the liposome by ultraviolet-visible spectrophotometry; (2T-P: 292 nm; 2T-N: 285 nm). PLL was prepared at various concentrations from 0 mol% to 50 mol%. For samples below 50 mol%, the amount of lipid remained equal. Each sample was extruded a total of 11 times through a 200nm pore membrane. Liposomes before and after extrusion were broken by dissolution in DMSO (liposome suspension/DMSO, 1:9, v/v). The samples were analyzed in quartz cuvettes. The following parameters were used: scanning speed: 120 nm/min; bandwidth: 2 nm; integration time: 0.15 sec; data interval: 0.30 nm; initial wavelength: 500 nm; end wavelength: 190 nm.

N and P (scheme 1) are hydrophobic compounds that have been modified to contain amphiphilic tails (2T-P and 2T-N) to alter their incorporation in liposomes, which can be characterized using uv-visualization. This technique measures the transmittance that is reduced through the surface of the sample. Absorption of liposomes at different concentrations (after removal of unincorporated material) can be used to calculate how much drug has been incorporated into the liposomes.

Figure 7 panel a shows drug incorporation in liposomes before and after extrusion for 2T-P. Figure 7 panel B shows drug incorporation in liposomes before and after extrusion for 2T-N. For the parent compound, the liposomal drug incorporation limit was calculated to be < 10 mol%, and for the prodrug about 40 mol%. The maximum measured loading of the parent compound is very low (e.g., about 11 mol% for P and about 1.1 mol% for N).

The liposome solution is passed through a membrane to remove unbound molecules. Samples were analyzed before and after extrusion. Both prodrugs achieved high loading capacity, with 2T-N maintaining 96% of the loading capacity (fig. 6). At above 50 mol%, incorporation cannot be achieved because further extrusion is not possible. High standard deviation indicates instability of the liposomes due to unincorporated drug molecules within the liposomes.

The size distribution of the liposomes loaded with the parent compound and prodrug was monitored over time using Dynamic Light Scattering (DLS). Figure 8 panel a shows that 2T-P loaded liposomes remained stable for the entire 3 week period. Figure 8 panel B shows that P-loaded liposomes were stable over the entire 3 week period. Figure 8 panel C shows that 2T-N loaded liposomes remained stable for the entire 3 week period. Figure 8 panel D shows that N-loaded liposomes remained stable for the entire 3 week period. Conjugation of potent drugs to the phospholipid tail results in high loading efficiency, stability, retention and targeted delivery of liposomes.

The concentration of P-loaded and N-loaded liposomes reflects the initial drug amount, rather than the final drug retention after extrusion, which is no more than 15 mol% (figure 7 panel a and figure 7 panel B).

The size distribution of the prodrug-loaded liposomes indicates that the prodrug remains in the liposome with minimal leakage. Complete saturation was observed when extrusion became impossible due to the presence of excess unincorporated drug particles. Both increased loading efficiency and encapsulation stability can minimize chemical degradation and allow the use of viable prodrugs in lipophilic vehicles. Solubility is increased by adding a hydrophilic tail and converting the hydrophobic drug into a lipophilic prodrug. The conversion results in high entrapment efficiency, low leakage and chemical stability.

Example 8: 2T-T incorporation in liposomes

2T-T liposomes having an increased concentration of 2T-T are produced and passed through an extruder to remove any unbound 2T-T from the liposome solution. The liposome solution was analyzed before and after extrusion using uv-vis spectroscopy with a scan range of 200nm-500nm, 2nm bandwidth, 30 second integration time, 0.5nm data interval and 100nm/min scan speed. As can be seen in fig. 9 and table 1, the amount of 2T-T present after extrusion was similar to the extruded prodrug concentration, indicating minimal prodrug loss. Incorporation limits were analyzed, up to 70 mol% (147 uM).

The size distribution of the 2T-T liposomes was characterized by DLS measurements before and after extrusion. The standard deviation of liposome diameter was lower after extrusion. The results indicate an increase in the monodisperse population of liposomes. The size distribution of 2T-T liposomes loaded with various concentrations of 2T-T can be seen in Table 2 and FIGS. 10-11.

TABLE 1

TABLE 2

2T-T mol％	Front side	PDI	Rear end	PDI
						0	148±79	0.3±0.1	122±7	0.3±0
10	102±19	0.3±0	130±7	0.3±0
					20	121±16	0.3±0.1	129±9	0.3±0
30	98±28	0.3±0	118±25	0.3±0
					40	114±24	0.3±0	123±10	0.3±0
50*	122.4	0.3	122.4	0.3
					60*	182.9	0.5	88.76	0.3
70*	172.1	0.4	75.43	0.3

PDI ═ polydispersity index

Example 9: 2T-C liposome size distribution

2T-C liposomes having an increased concentration of 2T-C are produced and passed through an extruder to remove any unbound 2T-C from the liposome solution. The size distribution of the 2T-C liposomes was characterized by DLS measurements before and after extrusion. The standard deviation of liposome diameter was lower after extrusion. The results indicate an increase in the monodisperse population of liposomes. The size distribution of 2T-C liposomes loaded with various concentrations of 2T-C can be seen in Table 3 and FIGS. 12-13.

TABLE 3

2T-C mol％	Front side	PDI	Rear end	PDI
						0	117±7	0.3±0	77±13	0.3±0
10	124±11	0.3±0	121±8	0.3±0
					20	115±14	0.3±0	116±1	0.3±0
30	105±4	0.3±0	120±12	0.3±0
					40	109±15	0.3±0	115±7	0.3±0
50	76.6±13	0.4±0.1	91±2	0.3±0

PDI ═ polydispersity index

Example 10: cell culture

Human cervical cancer (ATCC S3) (HeLa) cells were cultured in DMEM supplemented with 10% FBS, 100mg/L penicillin G and 100mg/L streptomycin. Human breast cancer (MCF-7) cells were cultured in DMEM supplemented with 1.0mM sodium pyruvate, 1% GlutaMax-1, 100. mu.g/mL penicillin, 100. mu.g/mL streptomycin, and 10% FBS. Cells were incubated in 5% CO₂At 37 ℃. MCF10A cells were cultured in RPMI supplemented with 5% FBS, epidermal growth factor (20ng/mL), hydrocortisone (0.5ug/mL), cholera toxin (100ng/mL), insulin (10ug/mL) and PenStrep.

Example 11: in vitro cytotoxicity of liposomes loaded with 2T-P and 2T-N prodrugs

Cells were seeded at 4,000 cells/well (HeLa, MCF7, MCF10a) in 96-well plates and incubated at 37 ℃ and 5% CO₂Incubate for 24 hours. After incubation, the medium was changed and the cells were divided in parallel into PLL-treated and parent compound-treated groups. Cells were treated with a two-fold dilution series of PLL or 1 μ M of parent compound starting from a 2 vol% suspension of PLL (prodrug/lipid, 0.2 mol%). PLL group sunThe sexual control was as follows: untreated, empty liposomes (no drug, 2 vol%) and phenylarsonic oxide (PAO). The parent compound group positive controls were as follows: untreated, DMSO and PAO. After 48 hours of incubation, 20. mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT,5mg/mL) was added to each well and the wells were incubated for 2 hours. The medium was replaced with 100 μ L of DMSO to dissolve formazan crystals. The absorbance at 595nm was measured using a microplate reader. Experiments were performed in quadruplicate and repeated twice.

The cytotoxicity of prodrug-loaded liposomes (20 mol%) and the parent compound was examined using the standard MTT assay for HeLa, MCF-7 and MCF10A cell lines. 2T-N loaded liposomes retain potency, while 2T-P loaded liposomes lose activity. The reduced activity is due to steric hindrance within the 2T-P liposome structure. As a control, normal breast cancer cell lines were studied to verify cancer cell differentiation when cells were exposed to prodrug-loaded liposomes and parent compound-loaded liposomes. The toxicity of 2T-P loaded liposomes was significantly reduced in all cell lines tested compared to the parent compound P. 2T-N loaded liposomes maintained potency in HeLa cells (0.020 μ M), but showed reduced potency in MCF7(0.038 μ M). The efficacy of 2T-N loaded liposomes in MCF10A cells was significantly reduced (1.105. mu.M). Table 4 shows cell viability data for HeLa, MCF-7 and MCF10A cell lines after 48 hours of treatment with either the free parent compound or the prodrug-incorporating liposomes.

TABLE 4

Example 12: in vitro cytotoxicity of liposomes loaded with 2T-T and 2T-C prodrugs

To assess the cytotoxicity of 2T-T prodrug-loaded liposomes, HeLa cells were exposed to different concentrations of topotecan or 2T-T loaded liposomes for 72 hours. After a 72 hour incubation period, cell viability was assessed by MTT assay. As can be seen in fig. 14, IC of free topotecan was found₅₀51. + -.1 nM, and IC of 2T-T liposomes₅₀263. + -.430 nM.

To assess the cytotoxicity of 2T-C prodrug-loaded liposomes, HeLa cells were exposed to varying concentrations of cytarabine or 2T-C (NH) loaded liposomes₂Attached) for 48 hours in liposomes. After a 48 hour incubation period, cell viability was assessed by MTT assay. As can be seen in FIG. 15, the IC of free cytarabine was found₅₀4.45. + -. 6.29. mu.M, and IC of 2T-C liposomes₅₀It was 48.54. + -. 17.89. mu.M.

Example 13: in vitro ultrasound

Microbubbles were generated at 0 mol% and 20 mol% prodrug concentration purified by centrifugation at 0.3rcf for 10 minutes. The supernatant (microbubbles) and liposomes were isolated. The supernatant was incubated in serum-containing medium for 12 hours, and then centrifuged again. HeLa cells were seeded at 120,000 cells/well onto 6-well plate coverslips. After fusion of cells was observed, the coverslip was mounted into a cell plate chamber in contact with 2. mu.L of microvesicle 3mL of culture medium. The chamber was sealed with another coverslip, placed in an ultrasound chamber, and exposed to 18 ultrasound pulses. The coverslip was then immediately washed 3 times with medium to remove excess microbubbles. After 20 hours of incubation, the cell plates were imaged in a bright field using a microscope. Untreated as a control, inverted cell plates were used to facilitate contact with microbubbles loaded with 0 mol% and 20 mol% prodrug without ultrasound exposure.

Prodrugs are incorporated into microbubbles for focused drug release using ultrasound. Figure 16 shows in vitro ultrasound delivery of prodrug-loaded microbubbles. Microvesicles were purified, incubated in serum, and then purified again prior to testing. The microvesicles were not dissociated from the serum. In vitro cytotoxicity of local microbubble delivery using ultrasound validated local and ultrasound-triggered delivery of 2T-N infused microbubbles. Empty microbubbles and prodrug-loaded microbubbles were contacted with HeLa cells for ultrasound-free control to confirm localization using ultrasound (fig. 16, left panel). The empty and 2T-P loaded microbubbles remain fused with the sonicated cells in both the ultrasound exposed and ultrasound unexposed areas. 2T-N loaded microbubbles decrease with sonicated cells in the ultrasound-exposed regions, but remain fused in the ultrasound-unexposed regions.

Example 14: enzymatic assay

Enzymatic cleavage was measured qualitatively using a spectrofluorometer. Pig liver esterase was diluted to 1.2X10 from concentrated stock in 1X PBS^-7M, and storing at-20 ℃. At time zero, 100. mu.L of empty liposomes or PLL were placed in a 100. mu.L cuvette and 5. mu.L of esterase was added. The solution was immediately measured in a fluorescence spectrophotometer. The sample was measured again at 60 minutes. The following parameters were used: medium scanning speed, 2 seconds response time, 1nm sampling interval, 3nm excitation slit width, 20nm emission slit width, high sensitivity, 250nm excitation wavelength and 280nm-600nm emission range.

FIG. 17 shows the fluorescence spectra of 2T-N loaded liposomes at different time points with and without treatment with pig liver esterase. Table 5 shows the size change of the empty liposomes and 2T-N liposomes after esterase treatment. FIG. 18 shows the fluorescence spectra of empty liposomes at different time points with and without treatment with pig liver esterase. FIG. 19 shows fluorescence spectra of PBS solutions at various time points with and without treatment with pig liver esterase.

TABLE 5

Detailed description of the preferred embodiments

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1. a lipid-based carrier comprising: a) a surface layer, wherein the surface layer comprises a prodrug, wherein the prodrug comprises a therapeutic agent covalently conjugated to a phospholipid; and b) a core, wherein the surface layer surrounds the core.

Embodiment 2. the lipid-based carrier of embodiment 1, wherein the lipid-based carrier is a microvesicle.

Embodiment 3. the lipid-based carrier according to

embodiment

1 or 2, wherein the surface layer is a lipid monolayer.

Embodiment 4. the lipid-based carrier according to any one of embodiments 1-3, wherein the core is a gas.

Embodiment 5. the lipid-based carrier of embodiment 4, wherein the gas is sulfur hexafluoride (SF)₆)。

Embodiment 6. the lipid-based carrier according to embodiment 1 or 3, wherein the core is a solid.

Embodiment 7. the lipid-based carrier according to embodiment 6, wherein the solid is a metal.

Embodiment 8 the lipid-based carrier of embodiment 6, wherein the solid is a semiconductor.

Embodiment 9. the lipid-based carrier according to embodiment 1 or 3, wherein the core is a liquid.

Embodiment 10. the lipid-based carrier according to any one of embodiments 1-3, wherein the core is an organic material.

Embodiment 11 the lipid-based carrier according to any one of embodiments 1-3, wherein the core is an inorganic material.

Embodiment 12. the lipid-based carrier according to embodiment 1 or 3, wherein the core is an aqueous solution.

Embodiment 13. the lipid-based carrier according to any one of embodiments 1 or 4-12, wherein the lipid-based carrier is a liposome.

Embodiment 14. the lipid-based carrier according to any one of

embodiments

1,2 or 4 to 13, wherein the surface layer is a lipid bilayer.

Embodiment 15. the lipid-based carrier according to any one of embodiments 1-14, wherein the lipid-based carrier has a diameter of about 70nm to about 900 nm.

Embodiment 16. the lipid-based carrier of any one of embodiments 1-15, wherein the prodrug is present in an amount of about 1 mol% to about 100 mol%.

Embodiment 17. the lipid-based carrier according to any one of embodiments 1-16, wherein the phospholipid is a di-tailed phospholipid.

Embodiment 18 the lipid-based carrier of any one of embodiments 1-17, wherein the phospholipid comprises a hydrophobic tail comprising from about 10 carbon atoms to about 24 carbon atoms.

Embodiment 19. the lipid-based carrier of any one of embodiments 1-18, wherein the phospholipid comprises a hydrophobic tail comprising about 16 carbon atoms.

Embodiment 20 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is an anti-cancer agent.

Embodiment 21 the lipid-based carrier of embodiment 20, wherein the anticancer agent is topotecan.

Embodiment 22 the lipid-based carrier of embodiment 20, wherein the anti-cancer agent is cytarabine.

Embodiment 23. the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a compound of the formula:

embodiment 24. the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a compound of the formula:

embodiment 25 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is an antiviral agent.

Embodiment 26 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is an antibacterial agent.

Embodiment 27. the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a neurotransmitter.

Embodiment 28. the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a protein.

Embodiment 29 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a biologic.

Embodiment 30 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is gemcitabine.

Embodiment 31 the lipid-based carrier of any one of embodiments 1-19, wherein the therapeutic agent is a chelator.

Embodiment 32. the lipid-based carrier according to any one of embodiments 1-31, wherein the surface layer further comprises DPPC.

Embodiment 33 the lipid-based carrier according to any one of embodiments 1-32, wherein the surface layer further comprises DPPA.

Embodiment 34 the lipid-based carrier according to any one of embodiments 1-33, wherein the surface layer further comprises DSPE-PEG 2000.

Embodiment 35 the lipid-based carrier of any one of embodiments 1-34, wherein the surface layer further comprises DSPE-PEG 5000.

Embodiment 36. the lipid-based carrier according to any one of embodiments 1-35, wherein the phospholipid is an activated phospholipid.

Embodiment 37. the lipid-based carrier of embodiment 36, wherein the activated phospholipid is Glu-phospholipid.

Embodiment 38. the lipid-based carrier of embodiment 36, wherein the activated phospholipid is a NHS-phospholipid.

Embodiment 39. the lipid-based carrier of embodiment 36, wherein the activated phospholipid is a PDP-phospholipid.

Embodiment 40. the lipid-based carrier of embodiment 36, wherein the activated phospholipid is a MAL-phospholipid.

Embodiment 41. the lipid-based carrier of embodiment 36, wherein the activated phospholipid is NBD-phospholipid.

Embodiment 42. the lipid-based carrier of embodiment 37, wherein the Glu-phospholipid is DPPE-Glu.

Embodiment 43 the lipid-based carrier according to any one of embodiments 1-42, wherein the therapeutic agent is covalently conjugated to the phospholipid through an ester bond.

Embodiment 44 the lipid-based carrier according to any one of embodiments 1-42, wherein the therapeutic agent is covalently conjugated to the phospholipid through an amide bond.

Embodiment 45 a method of treating a disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the lipid-based carrier, the lipid-based carrier comprising: a) a surface layer, wherein the surface layer comprises a prodrug, wherein the prodrug comprises a therapeutic agent covalently conjugated to a phospholipid; and b) a core, wherein the surface layer surrounds the core.

Embodiment 46. the method of embodiment 45, wherein the lipid-based carrier is a microbubble.

Embodiment 47 the method of embodiment 45 or 46, wherein the surface layer is a lipid monolayer.

Embodiment 48 the method of any one of embodiments 45-47, wherein the core is a gas.

Embodiment 49 the method of embodiment 48, wherein the gas is sulfur hexafluoride (SF)₆)。

Embodiment 50 the method of embodiment 45 or 47, wherein the core is a solid.

Embodiment 51 the method of embodiment 50, wherein the solid is a metal.

Embodiment 52 the method of embodiment 50, wherein the solid is a semiconductor.

Embodiment 53 the method of embodiment 45 or 47, wherein the core is a liquid.

Embodiment 54 the method of any one of embodiments 45-47, wherein the core is an organic material.

Embodiment 55 the method of any one of embodiments 45-47, wherein the core is an inorganic material.

Embodiment 56 the method of embodiment 45 or 47, wherein the core is an aqueous solution.

Embodiment 57 the method of any one of embodiments 45 or 48-56, wherein the lipid-based carrier is a liposome.

Embodiment 58 the method of any one of embodiments 45, 46 or 48-57, wherein the surface layer is a lipid bilayer.

Embodiment 59. the method of any one of embodiments 45-58, wherein the lipid-based carrier has a diameter of about 70nm to about 900 nm.

Embodiment 60 the method of any one of embodiments 45-59, wherein the prodrug is present in an amount of about 1 mol% to about 100 mol%.

Embodiment 61 the method of any one of embodiments 45-60, wherein the phospholipid is a di-tailed phospholipid.

Embodiment 62. the method of any of embodiments 45-61, wherein the phospholipid comprises a hydrophobic tail comprising from about 10 carbon atoms to about 24 carbon atoms.

Embodiment 63. the method of any of embodiments 45-62, wherein the phospholipid comprises a hydrophobic tail comprising about 16 carbon atoms.

Embodiment 64 the method of any one of embodiments 45-63, wherein the therapeutic agent is an anti-cancer agent.

Embodiment 65 the method of embodiment 64, wherein the anti-cancer agent is topotecan.

Embodiment 66 the method of embodiment 64, wherein the anti-cancer agent is cytarabine.

Embodiment 67. the method of any one of embodiments 45-63, wherein the therapeutic agent is a compound of the formula:

embodiment 68 the method of any one of embodiments 45-63, wherein the therapeutic agent is a compound of the formula:

embodiment 69 the method of any one of embodiments 45-63, wherein the therapeutic agent is an antiviral agent.

Embodiment 70 the method of any one of embodiments 45-63, wherein the therapeutic agent is an antibacterial agent.

Embodiment 71 the method of any one of embodiments 45-63, wherein the therapeutic agent is a neurotransmitter.

Embodiment 72 the method of any one of embodiments 45-63, wherein the therapeutic agent is a protein.

Embodiment 73. the method of any one of embodiments 45-63, wherein the therapeutic agent is a biologic.

Embodiment 74 the method of any one of embodiments 45-63, wherein the therapeutic agent is gemcitabine.

Embodiment 75. the method of any one of embodiments 45-63, wherein the therapeutic agent is a chelator.

Embodiment 76 the method of any one of embodiments 45-75, wherein the surface layer further comprises DPPC.

Embodiment 77 the method of any one of embodiments 45-76, wherein the surface layer further comprises DPPA.

Embodiment 78 the method of any one of embodiments 45-77, wherein the surface layer further comprises DSPE-PEG 2000.

Embodiment 79 the method of any one of embodiments 45-78, wherein the surface layer further comprises DSPE-PEG 5000.

Embodiment 80. the method of any one of embodiments 45-79, wherein the phospholipid is an activated phospholipid.

Embodiment 81. the method of embodiment 80, wherein the activated phospholipid is Glu-phospholipid.

Embodiment 82 the method of embodiment 80, wherein the activated phospholipid is an NHS-phospholipid.

Embodiment 83 the method of embodiment 80, wherein the activated phospholipid is a PDP-phospholipid.

Embodiment 84. the method of embodiment 80, wherein the activated phospholipid is a MAL-phospholipid.

Embodiment 85 the method of embodiment 80, wherein the activated phospholipid is NBD-phospholipid.

Embodiment 86. the method of embodiment 81, wherein the Glu-phospholipid is DPPE-Glu.

Embodiment 87. the method of any one of embodiments 45-86, wherein the therapeutic agent is covalently conjugated to the phospholipid through an ester bond.

Embodiment 88. the method of any one of embodiments 45-86, wherein the therapeutic agent is covalently conjugated to the phospholipid through an amide bond.

Embodiment 89 the method of any one of embodiments 45-89, further comprising applying an in vitro trigger to the subject.

Embodiment 90 the method of embodiment 89, wherein the in vitro trigger is an ultrasound frequency.

Embodiment 91 the method of embodiment 90, wherein the ultrasound frequency is from about 1MHz to about 20 MHz.

Embodiment 92 the method of embodiment 89, wherein the in vitro trigger is light.

Embodiment 93 the method of embodiment 92, wherein the light has a wavelength of about 400nm to about 1400 nm.

Embodiment 94 the method of embodiment 89, wherein said in vitro trigger is an electric field.

Embodiment 95 the method of embodiment 89, wherein the in vitro trigger is a magnetic field.

Embodiment 96 the method of embodiment 95, wherein the magnetic field has a strength of about 0.2T to about 7T.

Embodiment 97 the method of any one of embodiments 89-96, wherein the in vitro trigger is applied in pulses.

Embodiment 98 the method of any one of embodiments 45-97, wherein the administering is intravenous.

Embodiment 99 the method of any one of embodiments 45-97, wherein said administering is intratumoral.

Embodiment 100 the method of any one of embodiments 45-97, wherein the administering is subcutaneous.

Embodiment 101 the method of any one of embodiments 45-97, wherein the administering is intraarterial.

Claims

1. A lipid-based carrier comprising:

a) a surface layer, wherein the surface layer comprises a prodrug, wherein the prodrug comprises a therapeutic agent covalently conjugated to a phospholipid; and

b) a core, wherein the surface layer surrounds the core.

2. The lipid-based carrier of claim 1, wherein the lipid-based carrier is a microbubble.

3. The lipid-based carrier of claim 1, wherein the surface layer is a lipid monolayer.

4. The lipid-based carrier of claim 1, wherein the core is a gas.

5. The lipid-based carrier of claim 4, wherein the gas is sulfur hexafluoride (SF)₆)。

6. The lipid-based carrier of claim 1, wherein the core is a solid.

7. The lipid-based carrier of claim 6, wherein the solid is a metal.

8. The lipid-based carrier of claim 6, wherein the solid is a semiconductor.

9. The lipid-based carrier of claim 1, wherein the core is a liquid.

10. The lipid-based carrier of claim 1, wherein the core is an organic material.

11. The lipid-based carrier of claim 1, wherein the core is an inorganic material.

12. The lipid-based carrier of claim 1, wherein the core is an aqueous solution.

13. The lipid-based carrier of claim 1, wherein the lipid-based carrier is a liposome.

14. The lipid-based carrier of claim 1, wherein the surface layer is a lipid bilayer.

15. The lipid-based carrier of claim 1, wherein the lipid-based carrier has a diameter of about 70nm to about 900 nm.

16. The lipid-based carrier of claim 1, wherein the prodrug is present in an amount of about 1 mol% to about 100 mol%.

17. The lipid-based carrier of claim 1, wherein the phospholipid is a di-tailed phospholipid.

18. The lipid-based carrier of claim 1, wherein the phospholipid comprises a hydrophobic tail comprising from about 10 carbon atoms to about 24 carbon atoms.

19. The lipid-based carrier of claim 1, wherein the phospholipid comprises a hydrophobic tail comprising about 16 carbon atoms.

20. The lipid-based carrier of claim 1, wherein the therapeutic agent is an anticancer agent.

21. The lipid-based carrier of claim 20, wherein the anticancer agent is topotecan.

22. The lipid-based carrier of claim 20, wherein the anti-cancer agent is cytarabine.

23. The lipid-based carrier of claim 1, wherein the therapeutic agent is a compound of the formula:

24. the lipid-based carrier of claim 1, wherein the therapeutic agent is a compound of the formula:

25. the lipid-based carrier of claim 1, wherein the therapeutic agent is an antiviral agent.

26. The lipid-based carrier of claim 1, wherein the therapeutic agent is an antibacterial agent.

27. The lipid-based carrier of claim 1, wherein the therapeutic agent is a neurotransmitter.

28. The lipid-based carrier of claim 1, wherein the therapeutic agent is a protein.

29. The lipid-based carrier of claim 1, wherein the therapeutic agent is a biologic.

30. The lipid-based carrier of claim 1, wherein the therapeutic agent is gemcitabine.

31. The lipid-based carrier of claim 1, wherein the therapeutic agent is a chelator.

32. The lipid-based carrier of claim 1, wherein the surface layer further comprises DPPC.

33. The lipid-based carrier of claim 1, wherein the surface layer further comprises DPPA.

34. The lipid-based carrier of claim 1, wherein the surface layer further comprises DSPE-PEG 2000.

35. The lipid-based carrier of claim 1, wherein the surface layer further comprises DSPE-PEG 5000.

36. The lipid-based carrier of claim 1, wherein the phospholipid is an activated phospholipid.

37. The lipid-based carrier of claim 36, wherein the activated phospholipid is Glu-phospholipid.

38. The lipid-based carrier of claim 36, wherein the activated phospholipid is a NHS-phospholipid.

39. The lipid-based carrier of claim 36, wherein the activated phospholipid is a PDP-phospholipid.

40. The lipid-based carrier of claim 36, wherein the activated phospholipid is a MAL-phospholipid.

41. The lipid-based carrier of claim 36, wherein the activated phospholipid is NBD-phospholipid.

42. The lipid-based carrier of claim 37, wherein the Glu-phospholipid is DPPE-Glu.

43. The lipid-based carrier of claim 1, wherein the therapeutic agent is covalently conjugated to the phospholipid through an ester bond.

44. The lipid-based carrier of claim 1, wherein the therapeutic agent is covalently conjugated to the phospholipid through an amide bond.

45. A method of treating a disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a lipid-based carrier comprising:

b) a core, wherein the surface layer surrounds the core.

46. The method of claim 45, wherein the lipid-based carrier is a microbubble.

47. The method of claim 45, wherein the surface layer is a lipid monolayer.

48. The method of claim 45, wherein the core is a gas.

49. The method of claim 48, wherein the gas is sulfur hexafluoride (SF)₆)。

50. The method of claim 45, wherein the core is a solid.

51. The method of claim 50, wherein the solid is a metal.

52. The method of claim 50, wherein the solid is a semiconductor.

53. The method of claim 45, wherein the core is a liquid.

54. The method of claim 45, wherein the core is an organic material.

55. The method of claim 45, wherein the core is an inorganic material.

56. The method of claim 45, wherein the core is an aqueous solution.

57. The method of claim 45, wherein the lipid-based carrier is a liposome.

58. The method of claim 45, wherein the surface layer is a lipid bilayer.

59. The method of claim 45, wherein the lipid-based carrier has a diameter of about 70nm to about 900 nm.

60. The method of claim 45, wherein the prodrug is present in an amount of about 1 mol% to about 100 mol%.

61. The method of claim 45, wherein the phospholipid is a di-tailed phospholipid.

62. The method of claim 45, wherein the phospholipid comprises a hydrophobic tail comprising from about 10 carbon atoms to about 24 carbon atoms.

63. The method of claim 45, wherein the phospholipid comprises a hydrophobic tail comprising about 16 carbon atoms.

64. The method of claim 45, wherein the therapeutic agent is an anti-cancer agent.

65. The method of claim 64, wherein the anti-cancer agent is topotecan.

66. The method of claim 64, wherein the anti-cancer agent is cytarabine.

67. The method of claim 45, wherein the therapeutic agent is a compound of the formula:

68. the method of claim 45, wherein the therapeutic agent is a compound of the formula:

69. the method of claim 45, wherein the therapeutic agent is an antiviral agent.

70. The method of claim 45, wherein the therapeutic agent is an antibacterial agent.

71. The method of claim 45, wherein the therapeutic agent is a neurotransmitter.

72. The method of claim 45, wherein the therapeutic agent is a protein.

73. The method of claim 45, wherein the therapeutic agent is a biologic.

74. The method of claim 45, wherein the therapeutic agent is gemcitabine.

75. The method of claim 45, wherein the therapeutic agent is a chelator.

76. The method of claim 45, wherein the surface layer further comprises DPPC.

77. The method of claim 45, wherein the surface layer further comprises DPPA.

78. The method of claim 45, wherein the surface layer further comprises DSPE-PEG 2000.

79. The method of claim 45, wherein the surface layer further comprises DSPE-PEG 5000.

80. The method of claim 45, wherein the phospholipid is an activated phospholipid.

81. The method of claim 80, wherein the activated phospholipid is Glu-phospholipid.

82. The method of claim 80, wherein the activated phospholipid is a NHS-phospholipid.

83. The method of claim 80, wherein the activated phospholipid is a PDP-phospholipid.

84. The method of claim 80, wherein the activated phospholipid is a MAL-phospholipid.

85. The method of claim 80, wherein the activated phospholipid is NBD-phospholipid.

86. The method of claim 81, wherein said Glu-phospholipid is DPPE-Glu.

87. The method of claim 45, wherein the therapeutic agent is covalently conjugated to the phospholipid through an ester bond.

88. The method of claim 45, wherein the therapeutic agent is covalently conjugated to the phospholipid through an amide bond.

89. The method of claim 45, further comprising applying an in vitro trigger to the subject.

90. The method of claim 89, wherein the in vitro trigger is an ultrasound frequency.

91. The method of claim 90, wherein the ultrasound frequency is from about 1MHz to about 20 MHz.

92. The method of claim 89, wherein the in vitro trigger is light.

93. The method of claim 92, wherein the light has a wavelength of about 400nm to about 1400 nm.

94. The method of claim 89, wherein the in vitro trigger is an electric field.

95. The method of claim 89, wherein the in vitro trigger is a magnetic field.

96. The method of claim 95, wherein the magnetic field has a strength of about 0.2T to about 7T.

97. The method of claim 89, wherein the in vitro trigger is applied in pulses.

98. The method of claim 45, wherein the administration is intravenous.

99. The method of claim 45, wherein the administration is intratumoral.

100. The method of claim 45, wherein the administration is subcutaneous.

101. The method of claim 45, wherein the administration is intraarterial.